Compositions for the treatment and prevention of hoof and claw diseases

ABSTRACT

The present invention relates to a composition comprising antigenic material of keratinophilic fungi and/or keratinophilic yeasts for use in a method of treating and/or preventing hoof- and claw diseases in animals and a new  Trichophyton verrucosum  strain which can e.g. be used in such a method of treatment and/or prevention.

The present invention relates to a composition comprising antigenicmaterial of keratinophilic fungi and/or keratinophilic yeasts for use ina method of treating and/or preventing hoof- and claw diseases inanimals and a new Trichophyton verrucosum strain which can e.g. be usedin such a method of treatment and/or prevention.

Digital dermatitis (shortly DD, also called Mortellaro's disease orItalian foot rot), which was first described in 1974 by Cheli andMortellaro, is a big problem in dairy cows and beef cattle and ispresent on many dairy farms. DD is very infectious and difficult totreat and to prevent. Also, it is difficult to control the eliminationfrom the herd. This disease is causing significant economic lossesworldwide. Different kinds of bacteria are responsible for the skinlesions in the interdigital place, which are characterized by red or“strawberry like”, hairless and painful ulcer with epithelialhyperplasia and swelling at the affected sites. They are typicallylocated at the back of the foot, in the interdigital space. Inactive ortreated lesions may be very difficult to differentiate from other wartydigital lesions. DD should be differentiated from interdigitaldermatitis (ID), which results in necrosis or necrobacillosis of thedistal interdigital skin. Investigations identified bacterialspirochetes in sections of DD tissue. In further analysis many types ofbacteria were identified in DD lesions. In particular, Treponema sppsuch as T. phagedenis, T. vincentii, and T. denticola can develop theclinical sings of DD. A very strong complex of different etiologicalfactors was found.

Interdigital dermatitis (ID) usually occurs in dairy cattle and is oneof the main infectious causes of lameness. Exudative dermatitis, wet,and smelly erosions with crust or scab and sole ulcers characterize theclinical manifestation of skin lesions in the heel area and theinterdigital space. Also clinical symptoms on the dorsal surface of thedigits can be observed. The heels are painful.

Afterwards, hyperplasia (corns, fibroma) with chronic irritation of theinterdigital space is often observed resulting in the lameness. Amixture of different bacteria causes ID. Dichelobacter nodosus plays animportant role in the manifestation of clinical symptoms. Thismicroorganism is an anaerobe with strong photolytic properties. Mostly,the infection is caused by bacteria from infected cows wherein D.nodosus spreads from infected to non-infected animals. Although thebacteria cannot survive for long times in the ground they can persist inthe claws. The bacteria destroy the epidermis, but do not penetrate thedermal layers. The disintegration of the border between the skin and thesoft heel horn produces erosions and ulcers. The lesions causediscomfort.

Interdigital phlegmone (shortly IP, also called panaritium or foot rot)is a necrotic inflammation of the skin in the interdigital space.Manifestation of the disease is usually in an acute or subacute form.The lesions, erosion and injury in the interdigital skin serve asinfection atriums. Moreover, bacterial microflora may enter and infectthe subcutaneous tissue. Infected animals became lame, cellulitis andswelling. The clinical symptoms of IP are accompanied by a decrease inmilk production, decreased appetite and a fever. Mainly, themicroorganisms Dichelobacter nodosus, Staphylococcus aureus, Escherichiacoli, Arcanobacterium pyogenes, and other are involved in IP. The mainbacteria involved in IP is Fusobacterium necrophorum that is agram-negative, no spore forming, no flagellated, no motile, pleomorphicanaerobic bacteria. This bacterium produces a lipopolysaccharideendotoxin that has a necrotizing activity and causes necrosis of theskin and subcutaneous tissue.

DD, ID and IP, which are the most common infectious hoof and clawdiseases, are sporadically distributed worldwide but may be endemic inparticular in intensive beef or dairy cattle production units. Theincidence depends amongst other on weather, season of year, grazingperiods, and housing system. IP usually leads to lameness and to asignificant decrease in body weight, loss of fertility and decrease ofmilk production. The incidence can be between 5% and 30%. In the firstepidemic cases about 30% to 80% animals can show clinical sings of thedisease. However, on an average IP accounts only for up to 15% of theclaw diseases.

It was surprising that a lot of researchers suggest that the etiologicalfactors of DD, ID and IP are the same microorganisms, such asDichelobacter nodosus, Fusobacterium necroforun and Fusobacterium, sppwhich first destroy the epidermis and allow the spirochetes fromTreponema spp such as T. phagedenis, T. vincentii, and T. denticola togain entrance into deeper tissues for developing the clinical sings ofDD. Other bacterial species isolated from pathological material fromtissues affected with DD, ID and IP are Campylobacter spp,Staphylococcus aureus, Escherichia coli, Arcanobacterium pyogenes, andPrevotella spp. Also, it was suggested that a virus plays an importantrole in the pathogenesis of the diseases.

The typical treatment strategy for DD, ID and IP is the application ofantibiotics, antibacterial preparations and topical applications padswith antibiotics, antiseptics and astringent solutions. Intramuscularapplications of penicillin or oxitetracelline during 3 days show goodresults for the treatment of IP but are not effective in the treatmentof ID and DD.

For the treatment of IP, ID and DD the topical application of solubleoxytetracycline, lincomycin-spectinomycin, nitrofurazone or sulfapreparations, as well as a mixture of sulfamethazine powder andanhydrous copper sulfate can provide sufficient results. However, beforethe treatment the lesion must be thoroughly cleansed and the necrotictissue has to be removed.

For preventive use a footbath with 7 to 10% copper or zinc sulfate or 3to 5% formaldehyde solution can be used. Also footbaths withoxytetracycline or lincomycin-spectinomycin can be used. But, thesesolutions can contaminate the environment and are prohibited in someareas or countries. This method was accepted for control of IP and ID,but is less effective for the treatment of DD.

For treating DD, ID and IP a Treponema spp bacterin vaccine wasdeveloped in the US. It was shown that immunization with said vaccinecould reduce clinical symptoms of DD in cattle. But, it was found inclinical trials that herd-specific pathogens including Treponema sppcannot provide sufficient protection against DD. Cows given the bacterinduring the dry period had no reduction in lesions as compared withnon-vaccinated control animals. The same results were obtained inGermany. It was concluded that treatment is not likely to be aneffective therapeutic, control or prevention strategy for DD in thefuture. No respective commercial vaccine exists on the market.

A lot of investigations were done to induce protection against F.necrophorum by using different kinds of antigenic components. It wasreported that cattle injected with F. necrophorum culture supernatantcontaining leukotoxin had a low incidence of foot rot caused by F.necrophorum. The stimulation with supernatant of a high leukotoxinproducing strain of F. necrophorum, mixed with an adjuvant, resulted ina high antileukotoxin antibody titer when injected in steers andprovided significant protection to experimentally induced liverabscesses. F. necrophorum bacterin was used as an agent for immunizingcattle and sheep against liver necrosis. Moreover, cells from culture ofvirulent F. necrophorum isolates were inactivated with 0.4% formalin.Mice immunized with said inactivated cells and with live F. necrophorumhad no detectable bacteria in the liver, lung or spleen for up to 28days. It was concluded that immunization of mice with formalin-killed F.necrophorum conferred protection against infection. Also, the injectionof endotoxin with leukotoxic activity in immunized animals prevents theestablishment of F. necrophorum infection.

In order to produce such a leukotoxoid vaccine, the F. necrophorumbacteria was cultured in a way to enhance the elaboration of leukotoxinin the supernatant. Bacterial growth and leukotoxin release wasterminated, and a vaccine was prepared by inactivating at least theleukotoxin-containing supernatant. This was done by separating theleukotoxin-containing supernatant from the bacteria and subsequently byan inactivation with formalin, β-propiolactone, heat, radiation or anyother known method of inactivation. Alternatively, the entire culturecould be inactivated to form the vaccine.

Also known is another formalin-inactivated leukocidin-exotoxin obtainedfrom the production strain Fusobacterium necrophorum “0-1” (RussianResearch Institute of Experimental Veterinary Medicine) comprising 1-10%inactivated bacterial mass of said strain containing 7.0-7.7 billioncells per 1 cm, glycerol, a mixture of mineral oil “Markol-52”,emulsifier taken in weight ratios of 1:(0.9-1.1), a suspension of livespores of splenic fever vaccine strain 55 (Russian Research Institute ofVeterinary Virology and Microbiology) in saline, and adjuvant. Thus,immunization of the animals against F. necrophorum leukotoxin canprevent diseases associated with F. necrophorum infection, e.g., liverabscesses in cattle and sheep, and ID as well as IP in cattle.

A known vaccine for the prevention of necrobacteriosis in cattlecomprises endotoxin and exotoxin antigens derived from pathogensnecrobacteriosis (F. necrophorum), formalin inactivator, saline and anadjuvant based on mineral oil and lanolin. A known vaccine fornecrobacteriosis farm animals comprises a complex of soluble antigensfrom strains F. necrophorum I and II serotypes, formalin, latex andadsorbent adjuvant. However, the known vaccines contain antigens of onetype of pathogen and do not provide sufficient prophylactic activity fornecrobacillosis, a diseases with anaerobic microorganisms and purulentwound microflora.

Also known are at least several inactivated commercial vaccines for theprophylaxis and treatment of ID and IP. Known is for example apolyvalent vaccine for the prevention of necrobacteriosis in cattle.Said vaccine contains formalin-inactivated antigens of Fusobacteriumnecrophorum, Staph. aureus, Corynebac. pyogenes, Clostridium perfringenstype A toxoid Cl. perfringens type A, aluminium hydroxide adjuvant andimmunopotentiator GMDP(N-acetilglukozamenil-(1-4)-N-acetimuromil-Z-alanin-D-isoglutamine).Also known is the vaccine “Nekovak” which comprises inactivated culturesof Fusobacterium necrophorum VGNKI N “Kp-1” DEP and/or Fusobacteriumnecrophorum VGNKI N “Tula” DEP, Actinomyces (Corybacterium) pyogenesVGNKI No. 4/2334 DEP, Staphylococcus aureus VGNKI No. 7315 DEP andClostridium perfringens type A VGNKI No. 28 DEP. The vaccine alsocontains a toxoid from a strain of Clostridium perfringens type A VGNKINo. 28 DEP and aluminum hydroxide adjuvant. In addition, the vaccinecontains a salt solution, such as saline.

All known vaccines often fail to elicit a sufficient immune response andto protect the animals against interdigital dermatitis and interdigitalphlegmone. There are no effective vaccines against digital dermatitis.

Approaches concerning the use of inactivated dermatophytes asdermatomycosis vaccines are known in the art. For example, the activeimmunization against Trichophyton purpureum infection in rabbits with aninactivated suspension of Trichophyton rubrum hyphae is described. AlsoEP 393371 and WO 9307894 disclose inactivated dermatomycosis vaccinescomprising dermatophytes of the genus Trichophyton and/or Microsporum.Moreover, mycoses vaccines are described in WO 98/15284 that comprisehomogenized inactivated dermatophyte microconidia and inactivatedhomogenized yeast blastospores.

Dermatomycoses in animals are anthropozoonotic diseases of the skin andrelated tissue. Clinical symptoms are loss of hair in the affected area,hyperemia, scaling and asbestos-like scabs. Calves are more susceptibleto ringworm infection than older animals. Dermatomycoses are often alsocharacterized by localized infection of the skin. In cattle the lesionsare most commonly found on the neck, head and consist of grey-whitecrusts raised on the skin. In addition, dermatomycoses may result inalopecia. Transmission of ringworm-causing organisms from infectedanimals to people has been reported. Dermatomycoses in animals carry asubstantial socioeconomic impact. Diseased animals require prolongedtreatment and can spread the infection to both animals and humans. Uptill now, dermatomycoses have been treated using various types ofmedication applied locally to affected areas of the skin. These includeda number of ointments, liniments, solutions and other substancescontaining fungicides and fungistatic agents. The disadvantages of suchtreatments are that they are not very effective, they require theadoption of quarantine measures and disinfection of areas where animalshave been kept (rearing pens, vivaria, farms, zoos, circuses, etc.).Moreover, they require substantial funds to be spent on drugpreparations and veterinary treatment and they pose difficulties inimmobilizing the animals (for wild animals held in captivity).Inactivated vaccines were developed to treat trichophytosis in cattle,dermatophytosis in horses, cats and dogs as e.g. described in WO93/07894 and live vaccines to treat fur-bearing animals and rabbits (seeUSSR Patent No. 835446), camels (see USSR Patent No. 1190574) andothers. A vaccine had also been developed for the prevention andtreatment of mycosis in mammals (see WO 98/15284).

Thus, the object of the present invention is the provision of newcompositions for use in a method of treating and/or preventing hoof- andclaw diseases in animals. It is a further object of the presentinvention to provide compositions fur use in a method of treating and/orpreventing digital and/or interdigital dermatitis and/or interdigitalphlegmone in animals, in particular in bovidae and pigs. It is a furtherobject of the present invention to provide a new Trichophyton verrucosumstrain. It is a further object of the present invention to providecompositions fur use in a method of treating and/or preventingdermatophytosis.

These objects are solved by the subject matter defined in the claims.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the description may mean “one,” but itis also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

The term “about” means that the value stated, plus or minus 5% of thestated value, or the standard error for measurements of the given value,are contemplated.

The term “comprising” as used herein shall not be construed as beinglimited to the meaning “consisting of” (i.e. excluding the presence ofadditional other matter). Rather, “comprising” implies that optionallyadditional matter may be present. The term “comprising” encompasses asparticularly envisioned embodiments falling within its scope “consistingof” (i.e. excluding the presence of additional other matter) and“comprising but not consisting of” (i.e. requiring the presence ofadditional other matter), with the former being more preferred.

The term “hoof- and claw disease” as used herein refers in particular toinfectious hoof- and claw diseases in bovidae and/or pigs. Said diseasesare in particular caused by bacteria, fungi and/or viruses. Inparticular the term “hoof- and claw diseases” refers to digitaldermatitis, interdigital dermatitis and interdigital phlegmone.

The term “bovidae” as used herein refers in particular to cloven-hoofed,ruminant mammals including includes bison, African buffalo, waterbuffalo, antelopes, gazelles, sheep, goats, muskoxen, and cattle.

The term “lameness” as used herein refers in particular to lameness as aresult of an infection and damage to tissue. In particular, the term“lameness” refers to lameness due to hoof and claw diseases, moreparticularly due to digital dermatis, interdigital dermatitis andinterdigital phlegmone.

The term “chitosan” as used herein refers to a copolymer of2-amino-2-deoxy-D-Glucopyranose and 2-acetamido-2-deoxy-D-glucopyranose,where the degree of deacetylation is more than 50%, preferably more than60%, 70%, 80% or 90%. Chitosan may be chemically derived from chitinwhich is a poly-1,4-β-N-acetyl-D-glucosamine, more particularly aN-acetyl-1,4-β-D-glucopyranosamine by deacetylation. Typical chitosanpreparations have varying molecular weights depending on the method ofmanufacture.

It was now surprisingly found, that vaccines comprising antigenicmaterial of keratinophilic fungi or yeasts confer good resistance andprophylactic and curing effect against digital dermatitis, interdigitaldermatitis and interdigital phlegmon in animals. In particular, it wassurprisingly found that vaccines comprising homogenised inactivateddermatophyte microconidia and/or inactivated homogenized yeastblastospores confer good resistance and prophylactic and curing effectagainst digital dermatitis, interdigital dermatitis and interdigitalphlegmon in animals.

The present invention relates to a composition comprising antigenicmaterial of keratinophilic fungi or keratinophilic yeasts for use in amethod of treating and/or preventing hoof- and claw diseases in animals.Preferably, the animals are mammals, more preferably bovidae and/orpigs, most preferably cattle.

The antigenic material of keratinophilic fungi or yeasts may be derivedfrom any parts of keratinophilic fungi or yeasts comprising antigenssuch as from the mycelium, artrospores, dermatophyte microconidia, yeastblastospores or others. The antigens are preferably polysaccharidesand/or glycopeptides. Preferably, the antigenic material ofkeratinophilic fungi or yeasts is selected from the group consisting of:homogenised inactivated dermatophyte microconidia, homogenisedinactivated yeast blastospores, antigenic material of yeast blastosporesand antigenic material of dermatophyte microconidia. Thus, the presentinvention more particularly relates to a composition comprisinghomogenised inactivated dermatophyte microconidia and/or homogenisedinactivated yeast blastospores and/or antigenic material of yeastblastospores and/or dermatophyte microconidia.

In particular, the hoof- and claw diseases result in lameness. Moreparticularly, the hoof- and claw diseases are digital dermatitis and/orinterdigital dermatitis and/or interdigital phlegmone. The hoof- andclaw diseases and digital dermatitis, interdigital dermatitis,interdigital phlegmone, respectively, may be caused by Dichelobacternodosus, Fusobacterium necroforun, Fusobacterium spp, Treponema spp suchas T. phagedenis, T. vincentii, and T. denticola, Campylobacter spp,Staphylococcus aureus, Escherichia coli, Arcanobacterium pyogenes, andPrevotella spp. and/or a virus.

The antigenic material of keratinophilic yeasts, in particular the yeastblastospores, of the composition for use of the present invention belongpreferably to the genus Candida and more preferably to the speciesCandida albicans. The antigenic material of keratinophilic dermatophyte,in particular the dermatophyte microconidia, belong preferably to thegenera Trichophyton and/or Microsporum. More preferably, thedermatophyte microconidia belong to the species Trichophyton verrucosum,Trichophyton mentagrophytes, Trichophyton equinum, Trichophytonsarkisovii, Trichophyton rubrum, Trichophyton mentagrophytes,Microsporum canis and/or Microsporum gypseum. In particular, the speciesMicrosporum canis can be Microsporum canis var. obesum and/orMicrosporum canis var. distortum.

In a preferred embodiment of the present invention the yeastblastospores and the dermatophyte microconidia are obtained from strainsof the above mentioned species which have been obtained by directedselection based on spore production and/or attenuation. It is highlypreferred to use a strain which grow faster in nutrient medium, producesmore microconidia and blastospores, respectively, has a lower virulenceand/or no adverse reactions after its intramuscular application incomparison to any epizootic strain from which it is derived. Examples ofsuch strains are the strains Trichophyton mentagrophytes DSM-7279,Trichophyton verrucosum DSM-28406, Trichophyton rubrum DSM-9469,Trichophyton rubrum DSM-9470, Trichophyton rubrum DSM-9471, Trichophytonrubrum DSM-9472, Candida albicans DSM-9456, Candida albicans DSM-9457,Candida albicans DSM-9458, and Candida albicans DSM-9459. Thus, inespecially preferred embodiments of the present invention the yeastblastospores and the dermatophyte microconidia are obtained from strainsTrichophyton mentagrophytes DSM-7279, Trichophyton verrucosum DSM-28406,Trichophyton rubrum DSM-9469, Trichophyton rubrum DSM-9470, Trichophytonrubrum DSM-9471, Trichophyton rubrum DSM-9472, Candida albicansDSM-9456, Candida albicans DSM-9457, Candida albicans DSM-9458, andCandida albicans DSM-9459.

The strains Trichophyton rubrum DSM-9469, Trichophyton rubrum DSM-9470,Trichophyton rubrum DSM-9471, Trichophyton rubrum DSM-9472, Candidaalbicans DSM-9456, Candida albicans DSM-9457, Candida albicans DSM-9458,and Candida albicans DSM-9459 have been deposited according to theBudapest Treaty at the “Deutsche Sammlung von Mikroorganismen undZellkulturen” (DSM), Mascheroder Weg 1B, W-38124 Braunschweig, Germany(which current name and address is “Leibniz-Institut DSMZ-DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH” (DMSZ),InhoffenstraBe 7B, 38124 Braunschweig, GERMANY) on 5 Oct. 1994 by theBasotherm GmbH, Eichendorffweg 5, 88396 Biberach an der Riss. Thecurrent depositors of said strains are the applicants, namely Dr. IgorPolyakov and Dr.sc.Dr. Liudmila Ivanova, Eberhardtstr. 40, 89073 Ulm.

Trichophyton Rubrum, No. DSM-9469

The strain was deposited at the DSM on May 10, 1994 under Serial No.DSM-9469. The strain was obtained by directed selection based on sporeproduction and attenuation of the epizootic strain No. 533, which wasidentified on a skin of man in 1985. The strain was identified using the“Rebell-Taplin” key (Rebell, G., Taplin, D.: Dermatophytes, theirrecognition and identification, 3rd Print, University of Miami Press.Coral Gables, Fla., USA, 1978). The biological properties of the strainare described in Table A. Strain No. DSM-9469 differs from the epidemicstrain in its faster growth in nutrient medium, an enormous productionof microconidiae and lower virulence.

TABLE A Properties and characteristics of the strains Strain No.DSM-9469 Epidemic Strain No. 533 Description of the Mature 15-day colonyon agar 20-day colony on agar Sabouraud: culture Sabouraud: white,velvety, flat, white, downy, elevated, margin of margin of the colonyfringed, colony regular, under surface under surface yellow, in centrepurple, diameter of colony 30-35 mm deep purple, diameter of colony60-63 mm Morphological Mature 15-day culture with 20-day culture withseptate characteristics septate branching hyphae 1-3 μm branching hyphae1-3 μm wide, wide, numerous obovate microconidia clavate to round inoval microconidia measuring 2-3 × small open clusters and along the 3-5μm, macroconidia long hyphae measuring 2-3 × 3-6 μm; clavatepencil-shaped with 4-5 macroconidia are rare, long and cross wallsmeasuring 4-6 × pencil-shaped with 3-5 cross walls 15-40 μm. measuring4-7 × 15-50 μm. Pathogenic The strain is weakly virulent. 9-10 Thestrain is virulent. 9-10 days characteristics days after application ofa after application of a dose of 500-600 dose of 500-600 thousandthousand cells of fungal cells of fungal material per cm² material percm² on scarified skin on scarified skin of guinea pigs, of guinea pigs,thin necrotic scabs scales are formed. are formed. Spontaneous recoverySpontaneous recovery after 18-20 after 25-30 days. days. Reactionresponse Result of intramuscular injection Result of intramuscularinjection of of inactivated corpuscular inactivated corpuscular antigensantigens from cultures: no from cultures: inflammation at point observedchanges in clinical of injection, oedema state of animals ImmunogenicResults of immunisation of a Results of immunisation of a group responsegroup of guinea pigs with of guinea pigs with inactivated inactivatedantigen from antigen from cultures (repeated not cultures (repeated notless than less than 5 times): establishes 5 times): establishes immunityimmunityTrichophyton Rubrum, No. DSM-9470

The strain was deposited at the DSM on May 10, 1994 under Serial No.DSM-9470. The strain was obtained by directed selection based on sporeproduction and attenuation of the epizootic strain No. 535, which wasidentified on a skin of man in 1990. The strain was identified using the“Rebell-Taplin” key (Rebell, G., Taplin, D.: Dermatophytes, theirrecognition and identification, 3rd Print, University of Miami Press.Coral Gables, Fla., USA, 1978). The biological properties of the strainare described in Table B. Strain No. DSM-9470 differs from the epidemicstrain in its faster growth in nutrient medium, an enormous productionof microconidiae and lower virulence.

TABLE B Properties and characteristics of the strains Strain No.DSM-9470 Epidemic Strain No. 535 Description of the Mature 15-day colonyon 20-day colony on agar Sabouraud: culture agar Sabouraud: white white,fluffy, margin of colony velvety-fluffy in centre, regular, undersurface yellow, 20 mm folded, margin of colony in diameter regular,under surface colourless or rose, diameter of colony 25-30 mmMorphological Mature 15-day culture with 20-day culture with septatecharacteristics septate branching hyphae 1-3 μm branching hyphae 1-3 μmwide, wide, round oval microconidia clavate to round in puriformmicroconidia small open clusters and along the measuring 2-3 × 3-7 μm.hyphae measuring 2-3 × 3-6 μm; macroconidia are absent. Pathogenic Thestrain is weakly virulent. The strain is virulent. 9-10 dayscharacteristics 9-10 days after application after application of a doseof 500-600 of a dose of 500-600 thousand cells of fungal thousand cellsof fungal material per cm² on scarified skin of material per cm² onguinea pigs, thin necrotic scabs are scarified skin of guinea pigs,formed. Spontaneous recovery after necrotic scabs are formed. 25-30days. Spontaneous recovery after 22-25 days. Reaction response Result ofintramuscular Result of intramuscular injection of injection ofinactivated inactivated corpuscular antigens corpuscular antigens fromfrom cultures: inflammation at point cultures: no observed of injection,oedema changes in clinical state of animals Immunogenic Results ofimmunisation of a Results of immunisation of a group response group ofguinea pigs with of guinea pigs with inactivated inactivated antigenfrom antigen from cultures (repeated not cultures (repeated not lessless than 5 times): establishes than 5 times): establishes immunityimmunityTrichophyton Rubrum, No. DSM-9471

The strain was deposited at the DSM on May 10, 1994 under Serial No.DSM-9471. The strain was obtained by directed selection based on sporeproduction and attenuation of the epizootic strain No. 620, which wasidentified on a nail of man in 1989. The strain was identified using the“Rebell-Taplin” key (Rebell, G., Taplin, D.: Dermatophytes, theirrecognition and identification, 3rd Print, University of Miami Press.Coral Gables, Fla., USA, 1978). The biological properties of the strainare described in Table C. Strain No. DSM-9471 differs from the epidemicstrain in its faster growth in nutrient medium, an enormous productionof microconidiae and lower virulence.

TABLE C Properties and characteristics of the strains Strain No.DSM-9471 Epidemic Strain No. 620 Description of the Mature 15-day colonyon agar 20-day colony on agar culture Sabouraud: white, velvety,Sabouraud: white, downy, elevated, margin of colony elevated, margin ofcolony regular, under surface yellow, regular, under surface purple, incentre deep purple, diameter of colony 20-25 mm diameter of colony 32-35mm Morphological Mature 15-day culture with 20-day culture with septatecharacteristics septate branching hyphae 1-3 μm branching hyphae 1-3 μmwide, wide, round oval microconidia clavate to round in puriformmicroconidia small open clusters and along the measuring 2-3 × 3-7 μm.hyphae measuring 2-3 × 3-6 μm; macroconidia are rare, long andpencil-shaped with 3-5 cross walls measuring 4-7 × 15-50 μm. PathogenicThe strain is weakly virulent. 9-10 The strain is virulent. 9-10 dayscharacteristics days after application of after application of a dose of500-600 a dose of 500-600 thousand thousand cells of fungal cells offungal materials per materials per cm² on scarified cm² on scarifiedskin of skin of guinea pigs, thin necrotic guinea pigs, scales are scabsare formed. Spontaneous formed. Spontaneous recovery after 25-30 days.recovery after 18-20 days. Reaction response Result of intramuscularResult of intramuscular injection injection of inactivated ofinactivated corpuscular corpuscular antigens from antigens fromcultures: cultures: no observed inflammation at point of injection,changes in clinical state of oedema animals Immunogenic Results ofimmunisation of a Results of immunisation of a response group of guineapigs with group of guinea pigs with inactivated antigen from inactivatedantigen from cultures cultures (repeated not less (repeated not lessthan 5 times): than 5 times): establishes establishes immunity immunityTrichophyton Rubrum, No. DSM-9472

The strain was deposited at the DSM on May 10, 1994 under Serial No.DSM-9472. The strain was obtained by directed selection based on sporeproduction and attenuation of the epizootic strain No. 754, which wasidentified on a nail of man in 1990. The strain was identified using the“Rebell-Taplin” key (Rebell, G., Taplin, D.: Dermatophytes, theirrecognition and identification, 3rd Print, University of Miami Press.Coral Gables, Fla., USA, 1978). The biological properties of the strainare described in Table D. Strain No. DSM-9472 differs from the epidemicstrain in its faster growth in nutrient medium, an enormous productionof microconidiae and lower virulence.

TABLE D Properties and characteristics of the strains Strain No.DSM-9472 Epidemic Strain No. 754 Description of the Mature 15-day colonyon agar 20-day colony on agar culture Sabouraud: white, velvety, inSabouraud: white-rose, downy, centre folded, margin of margin of colonyregular, under colony regular, under surface surface purple, diameter ofcolony yellow in centre purple, 20-25 mm diameter of colony 35-40 mmMorphological Mature 15-day culture with 20-day culture with septatecharacteristics septate branching hyphae 1-3 μm branching hyphae 1-3 μmwide, wide, round oval microconidia clavate to round in puriformmicroconidia small open clusters and along the measuring 2-3 × 3-7 μm.hyphae measuring 2-3 × 3-6 μm; macroconidia are rare, long andpencil-shaped with 3-5 cross walls measuring 4-7 × 15-50 μm. PathogenicThe strain is weakly virulent. 9-10 The strain is virulent. 9-10 dayscharacteristics days after application of a after application of a doseof 500-600 dose of 500-600 thousand thousand cells of fungal cells offungal materials per materials per cm² on scarified cm² on scarifiedskin of skin of guinea pigs, thin necrotic guinea pigs, scales are scabsare formed. Spontaneous formed. Spontaneous recovery after 25-30 days.recovery after 18-20 days. Reaction response Result of intramuscularResult of intramuscular injection injection of inactivated ofinactivated corpuscular corpuscular antigens from antigens fromcultures: cultures: no observed inflammation at point of injection,changes in clinical state of oedema animals Immunogenic Results ofimmunisation of a Results of immunisation of a response group of guineapigs with group of guinea pigs with inactivated antigen from inactivatedantigen from cultures cultures (repeated not less (repeated not lessthan 5 times): than 5 times): establishes establishes immunity immunityCandida Albicans, No. DSM-9456

The strain was deposited at the DSM on May 10, 1994 under Serial No.DSM-9456. The strain was obtained by directed selection based onstabilisation of cultural-morphological characteristics and attenuationof epidemic strain No. 008-L, which was identified on man in 1990. Thestrain was identified using the Lodder's key (Lodder, J: The yeast: ATaxonomic Study. North-Holland Publ. Co., Amsterdam-London (1970). Thebiological properties of the strain are described in Table E. Strain No.DSM-9456 differs from the epidemic strain in its faster growth innutrient medium, stabile biological properties, an enormous productionof biomass and lower virulence.

TABLE E Properties and characteristics of the strains Strain No.DSM-9456 Epidemic Strain No. 008-L Description of the 10-daysingle-spore colony 10-day single-spore colony on culture on agarSabouraud: cream agar Sabouraud: cream soft and smooth and pastyglistening, smooth with feathery offshots at elevated, margin of colonythe edges, diameter of colony 10-15 mm regular, diameter of colony 20-30mm Morphological 10-day culture with spherical 10-day single-sporeculture on characteristics oval blastospores measuring agar Sabouraudwith spherical 3.5-6 × 6-10 μm, oval budding blastospores chiamidospores12-15 μm measuring 3-5 × 5-8 μm, wide, pseudohyphae 5-8 μmchlamidospores 10-15 μm wide, hyphae 1.5-3 μm diameter, pseudohyphae 5-8μm wide wide, hyphae 1.5-3 μm wide. Pathogenic The strain is weaklyvirulent. The strain is weakly virulent. 30 characteristics 30 daysafter intraperitoneal days after intraperitoneal injection injection ofa dose of 10-100 of a dose of 10-100 million million fungal cells tofungal cells to white mice, white mice, granuloma in granuloma inabdominal organs of abdominal organs of 50% of 80-100% of animals areformed. animals are formed. Lethal Lethal effect in 50-70% was effectwas not observed. observed. Reaction response Result of intramuscularResult of intramuscular injection injection of inactivated ofinactivated corpuscular corpuscular antigens from antigens fromcultures: cultures: no observed inflammation at point of injection,changes in clinical state of oedema animals Immunogenic Results ofimmunisation of a Results of immunisation of a response group of whitemice with group of white mice with inactivated antigen from inactivatedantigen from cultures cultures (repeated not less (repeated not lessthan 10 times): than 10 times): establishes establishes immunityimmunityCandida Albicans, No. DSM-9457 The strain was deposited at the DSM onMay 10, 1994 under Serial No. DSM-9457. The strain was obtained bydirected selection based on stabilisation of cultural-morphologicalcharacteristics and attenuation of epidemic strain No. 012, which wasidentified on man in 1992. The strain was identified using the Lodder'skey (Lodder, J: The yeast: A Taxonomic Study. North-Holland Publ. Co.,Amsterdam-London (1970). The biological properties of the strain aredescribed in Table F. Strain No. DSM-9457 differs from the epidemicstrain in its faster growth in nutrient medium, stabile biologicalproperties, an enormous production of biomass and lower virulence.

TABLE F Properties and characteristics of the strains Strain No.DSM-9457 Epidemic Strain No. 012 Description of the 10-day single-sporecolony 10-day single-spore colony on culture on agar Sabouraud: creamagar Sabouraud: cream rough rough elevated, margin of elevated, marginof colony fringed colony lobulated, diameter and lobulated, diameter ofcolony of colony 20-23 mm 15-20 mm Morphological 10-day single-sporeculture 10-day single-spore culture on characteristics with sphericaloval agar Sabouraud with spherical blastospores measuring 3.5-5 × ovalbudding blastospores 5-10 μm, measuring 3-5 × 5-8 μm, chlamidospores12-15 μm chlamidospores 10-15 μm wide, pseudohyphae 4-7 μm diameter,pseudohyphae 5-8 μm wide, hyphae 2-3 μm wide, hyphae-1.5-3 μm wide widePathogenic The strain is weakly virulent. The strain is weakly virulent.30 characteristics 30 days after intraperitoneal days afterintraperitoneal injection injection of a dose of 10-100 by dose of10-100 million fungal million fungal cells to cells to white mice,granuloma in white mice, granuloma in abdominal organs of 50% ofabdominal organs in 30% of animals are formed. Lethal effect animals areformed. Lethal not more 50% were observed. effect was not observed.Reaction response Result of intramuscular Result of intramuscularinjection injection of inactivated of inactivated corpuscularcorpuscular antigens from antigens from cultures: no cultures: noobserved observed changes in clinical state changes in clinical state ofof animals animals Immunogenic Results of immunisation of a Results ofimmunisation of a response group of white mice with group of white micewith inactivated antigen from inactivated antigen from cultures cultures(repeated not less (repeated not less than 10 times): than 10 times):establishes establishes immunity immunityCandida Albicans, No. DSM-9458

The strain was deposited at the DSM on May 10, 1994 under Serial No.DSM-9458. The strain was obtained by directed selection based onstabilisation of cultural-morphological characteristics and attenuationof epidemic strain No. 047, which was identified on man in 1989. Thestrain was identified using the Lodder's key (Lodder, J: The yeast: ATaxonomic Study. North-Holland Publ. Co., Amsterdam-London (1970). Thebiological properties of the strain are described in Table G. Strain No.DSM-9458 differs from the epidemic strain in its faster growth innutrient medium, stabile biological properties, an enormous productionof biomass and lower virulence.

TABLE G Properties and characteristics of the strains Strain No.DSM-9458 Epidemic Strain No. 047 Description of the 10-day single-sporecolony 10-day single-spore colony on culture on agar Sabouraud: creamagar Sabouraud: cream soft and smooth and pasty smooth with featheryoffshots at glistening, elevated, margin the edges, diameter of colony10-15 mm of colony regular, diameter of colony 16-18 mm Morphological10-day culture with spherical 10-day single-spore culture oncharacteristics oval blastospores measuring agar Sabouraud withspherical 3.6-6 × 6-11 μm, oval budding blastospores chlamidospores12-15 μm measuring 3-5 × 5-8 μm, wide, pseudohyphae 4-8 μmchlamidospores 10-15 μm wide, hyphae 1.5-3 μm diameter, pseudohyphae 5-8μm wide wide, hyphae 1.5-3 μm wide. Pathogenic The strain is weaklyvirulent. The strain is weakly virulent. 30 characteristics 30 daysafter intraperitoneal days after intraperitoneal injection injection ofa dose of 10-100 by dose of 10-100 million fungal million fungal cellsto cells to white mice, granuloma in white mice, granuloma in abdominalorgans of 80-100% of abdominal organs of 50-100% animals are formed.Lethal effect of animals are in 70-100% were observed. formed. Lethaleffect in 50% were observed. Reaction response Result of intramuscularResult of intramuscular injection injection of inactivated ofinactivated corpuscular corpuscular antigens from antigens fromcultures: cultures: no observed inflammation at point of injection,changes in clinical state of oedema animals Immunogenic Results ofimmunisation of a Results of immunisation of a response group of whitemice with group of white mice with inactivated antigen from inactivatedantigen from cultures cultures (repeated not less (repeated not lessthan 10 times): than 10 times): establishes establishes immunityimmunityCandida Albicans, No. DSM-9459

The strain was deposited at the DSM on May 10, 1994 under Serial No.DSM-9459. The strain was obtained by directed selection based onstabilisation of cultural-morphological characteristics and attenuationof epidemic strain No. 158, which was identified on man in 1990. Thestrain was identified using the Lodder's key (Lodder, J: The yeast: ATaxonomic Study. North-Holland Publ. Co., Amsterdam-London (1970). Thebiological properties of the strain are described in Table H. Strain No.DSM-9459 differs from the epidemic strain in its faster growth innutrient medium, stable biological properties, an enormous production ofbiomass and lower virulence.

TABLE H Properties and characteristics of the strains Strain No.DSM-9459 Epidemic Strain No. 158 Description of the 10-day single-sporecolony 10-day single-spore colony on culture on agar Sabouraud: creamagar Sabouraud: cream smooth smooth pasty glistening, pasty, margin ofcolony lobulated elevated, margin of colony and with feathery offshotsat the regular, diameter of colony edges, diameter of colony 10-15 mm16-18 mm Morphological 10-day culture with spherical 10-day single-sporeculture on characteristics oval blastospores measuring agar Sabouraudwith spherical 3.6-6 × 6-11 μm, oval budding blastospores chlamidospores12-15 μm measuring 3-5 × 5-8 μm, wide, pseudohyphae 4-8 μmchlamidospores 10-15 μm wide, hyphae 1.5-3 μm diameter, pseudohyphae 5-8μm wide wide, hyphae 1.5-3 μm wide. Pathogenic The strain is weaklyvirulent. The strain is weakly virulent. 30 characteristics 30 daysafter intraperitoneal days after intraperitoneal injection injection bydose of 10-100 by dose of 10-100 million fungal million fungal cells towhite cells to white mice, granuloma in mice, granuloma in abdominalorgans of 50% of abdominal organs of 40% of animals are formed. Lethaleffect animals are formed. Lethal in 20-50% was observed. effect was notobserved. Reaction response Result of intramuscular Result ofintramuscular injection injection of inactivated of inactivatedcorpuscular corpuscular antigens from antigens from cultures: cultures:no observed inflammation at point of injection, changes in clinicalstate of oedema animals Immunogenic Results of immunisation of a Resultsof immunisation of a response group of white mice with group of whitemice with inactivated antigen from inactivated antigen from culturescultures (repeated not less (repeated not less than 10 times): than 10times): establishes establishes immunity immunity

Strain Trichophyton mentagrophytes DSM-7279 has been deposited accordingto the Budapest Treaty at the “Deutsche Sammlung von Mikroorganismen andZellkulturen” (DSM), Mascheroder Weg 1B, W-38124 Braunschweig, Germany(which current name and address is “Leibniz-Institut DSMZ-DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH” (DMSZ),InhoffenstraBe 7B, 38124 Braunschweig, GERMANY) on 1 Oct. 1992 byBoehringer Ingelheim Vetmedica GmbH, 6507 Ingelheim am Rhein (whichcurrent address is Boehringer Ingelheim Vetmedica GmbH, 55216 Ingelheimam Rhein). The current depositors of said strain are the applicants,namely Dr. Igor Polyakov and Dr.sc.Dr. Liudmila Ivanova, Eberhardtstr.40, 89073 Ulm.

Trichophyton Mentagrophytes No. VKPGF-930/1032, No. DSM-7279

The strain was deposited at the DSM on 1 Oct. 1992 under Serial No.DSM-7279. The strain was obtained by directed selection based on sporeproduction and attenuation of the epizootic Strain No. 1032, which wasfound on a horse in 1985. The strain was identified as described aboveRebel, Taplin, loc. cit. and Kashkin, loc. cit.). The biologicalproperties are described in Table I. Strain No. VKPGF-930/1032,DSM-7279, differs from the epizootic strain by its faster growth innutrient medium, the enormous production of microconidia, its lowervirulence and the absence of any reaction with its antigens.

Trichophyton verrucosum, No. DSM-28406

The strain Trichophyton verrucosum BINO 348 was deposited by the BinomedGmbH (EinsteinstraBe 59, 89077 Ulm) according to the Budapest Treaty atthe—Leibniz-Institut DSMZ—Deutsche Sammlung von Microorganismen undZellkulturen GmbH, InhoffenstraBe 7B, 38124 Braunschweig, Germany underSerial No. DSM-28406 on 12 Feb. 2014. The depositor has authorized theapplicants to refer to the deposited biological material in theapplication and has given his unreserved and irrevocable consent to thedeposited material being made available to the public in accordance withRule 31 EPC. The strain was obtained by directed selection based onspore production and attenuation of epizootic strain Nr. 348, which wasisolated from cattle in 1997. The strain was identified using theRebell-Taplin key (Rebell, G., Taplin, D.: Dermatophytes, theirrecognition and identification, 1978) and according to Kashkin, P. N.et. al. (opredelitel patogennykh, toksigenykh vrednykh dlya chelovekagribov, 1979). The biological properties of the strain are described inTable J. Strain BINO 348-DSM 28406 differs from the epizootic strain inits faster growth in nutrient medium, the enormous production ofmicroconidia, lower virulence and the absence of any adverse reactionsafter intramuscular application of antigens.

TABLE I Properties and characteristics of strain Strain No.VKPGF-930/1032 Epizootic Strain No. 1032 Description of culture Mature10-15 day colony in agar/wort; Mature 25-30 day colony in agar/wort;cream, velvety/powdered, flat with white, flat, narrow, growing margin,slight flat elevation in center, narrow undersurface reddish-brown,colony growing margin, fringed, undersurface diameter: 15-20 mm lightbrown, colony diameter 25-30 mm Morphological characteristics Septate,branching hyphae 1-3 μm Septate, branching straight and spiral wide,numerous pyriform, oval hyphae 1-3 μm wide, round, flattenedmicroconidia measuring 1 to 3 × 2 to 6 μm, pyriform microconidiameasuring 1 to no macroconidia 3 × 2 to 6 μm, few elongate-ovalmacroconidia with 2-5 septates, measuring 2 to 6 × 15 to 25 μmPathogenic characteristics Necrotic scabs Dense, asbestos-like scabs 9to 10 days after application of a dose of 500-600 thousand cells offungal matter per cm² to the scarified skin of a rabbit Spontaneousrecovery after 22-25 days 30-35 days Reaction response No observedchanges in clinical state Inflammation at point of injection, Results ofsubcutaneous and intramuscular injection edema of inactivatedcorpuscular antigens from cultures Antigen response 20 to 25 days afterinjecting rabbits with corpuscular antigens, antibody titers observed inblood serum By PHR 1:320 to 1:640 1:320 to 1:640 By ELISA 1:400 to1:1600 1:400 to 1:1600 Immunogenic response Establishes immunityEstablishes immunity Immunization of a group of rabbits with inactivatedantigens from cultures (repeated at least 5 times)

TABLE J Properties and Strain No. DSM-28406 Epidemic Strain No. 348characteristics of the strains Description of the 20-day colony on MaltExtract 25-30-day colony in Malt Extract culture Agar: white orlight-yellow, Agar: light-yellow cream, velvety, velvety, furrowed,diameter of folded, undersurface colorless, colony 15-20 mm diameter ofcolony 10-12 mm Morphological Mature 20-day culture with Mature 25-30day culture with characteristics numerous oval, pyriform septatebranching mycelium, few microconidia measuring 1.5-3 × oval, pyriformmicroconidia 1 to 3 μm × 3-5 μm. 3 to 6 μm, macroconidia with 2 to 6septates, few arthrospores and chlamydospores 9-11 μm. Pathogenic Thestrain is weakly virulent. 9-10 The strain is virulent. 9-10 dayscharacteristics days after application of a after application of a doseof 500-600 dose of 500-600 thousand thousand cells of fungal cells offungal material per cm² material per cm² on scarified skin on scarifiedskin of guinea of guinea pigs, thin necrotic scabs pigs, scales areformed. are formed. Spontaneous recovery Spontaneous recovery after15-20 after 25-30 days. days. Reaction response Result of intramuscularResult of intramuscular injection injection of inactivated ofinactivated corpuscular antigens corpuscular antigens from fromcultures: inflammation at cultures: no observed changes point ofinjection, oedema in clinical state of animals Immunogenic Results ofimmunisation of a Results of immunisation of a group response group ofguinea pigs with of guinea pigs with inactivated inactivated antigenfrom antigen from cultures (repeated not cultures (repeated not lessthan less than 5 times): establishes 5 times): establishes immunityimmunity against dermatophytos against dermatophytos cause cause by T.verrucosum by T. verrucosum

In a preferred embodiment of the composition for use of the presentinvention the composition comprises homogenised inactivated dermatophytemicroconidia of one microconidia or a mixture of microconidia of two,three, four, five, six, seven, eight, nine or ten of the above listedstrains of dermatophytes. In a further preferred embodiment thecomposition comprises a mixture of homogenised inactivated dermatophytemicroconidia of one, two, three, four, five, six, seven, eight, nine orten of the above listed dermatophytes and homogenised inactivated yeastblastospores of one, two, three, or four of the above listed yeasts. Ina further preferred embodiment the composition comprises homogenisedinactivated dermatophyte microconidia of one or a mixture of two, three,or four of the above listed yeasts. The compositions may additionallycomprise antigenic material of dermatophyte microconidia and/orantigenic material of yeast blastospores as described herein.Alternatively or in addition, the compositions may additionally comprisechitosan modified by an organic carboxylic acid, or a salt thereof.

In a further preferred embodiment the composition comprises antigenicmaterial of one dermatophyte microconidia or a mixture of antigenicmaterial of dermatophyte microconidia of two, three, four, five, six,seven, eight, nine or ten of the above listed strains of dermatophyte.In a further preferred embodiment the composition comprises a mixture ofantigenic material of dermatophyte microconidia of one, two, three,four, five, six, seven, eight, nine or ten of the above listeddermatophytes and antigenic material of yeast blastospores of one, two,three, or four of the above listed yeasts. In a further preferredembodiment the composition comprises antigenic material of yeastblastospores of one or a mixture of two, three, or four of the abovelisted yeasts. The compositions may additionally comprise homogenisedinactivated dermatophyte microconidia of one, two, three, four, five,six, seven, eight, nine or ten of the above listed dermatophytes and/orhomogenised inactivated yeast blastospores of one, two, three, or fourof the above listed yeasts. Alternatively or in addition, thecompositions may additionally comprise chitosan modified by an organiccarboxylic acid, or a salt thereof.

In a preferred embodiment the composition for use of the presentinvention comprises a mixture of homogenised inactivated dermatophytemicroconidia of Trichophyton mentagrophytes, Trichophyton verrucosum,Trichophyton equinum, Trichophyton sarkisovii, Microsporum canis,Microsporum canis var. obesum, Microsporum canis var. distortum andMicrosporum gypseum. For example, the vaccine Polivac-TM (manufacturer:“Vetbiochim” LLC, Moscow; Distributor: “Prostore” LLC, Moscow) is inaccordance with this embodiment and can be used as a composition for useof the present invention. Polivac-TM is a vaccine designed for animalssuch as cats, dogs, horses and others. The composition may additionallycomprise antigenic material as described herein and/or chitosan modifiedby an organic carboxylic acid, or a salt thereof.

In a further preferred embodiment the composition for use of the presentinvention comprises a mixture of homogenised inactivated dermatophytemicroconidia of Trichophyton mentagrophytes, Trichophyton verrucosum andTrichophyton sarkisovii. For example, the vaccine Polivac-T(manufacturer: “Vetbiochim” LLC, Moscow; Distributor: “Prostore” LLC,Moscow) is in accordance with this embodiment and can be used as acomposition for use of the present invention. Polivac-T is a vaccinespecifically designed for cattle. The composition may additionallycomprise antigenic material as described herein and/or chitosan modifiedby an organic carboxylic acid, or a salt thereof. In a further preferredembodiment the composition for use of the present invention comprises amixture of homogenised inactivated dermatophyte microconidia ofTrichophyton mentagrophytes, Trichophyton verrucosum and Trichophytonsarkisovii and homogenized inactivated yeast blastospores of Candidaalbicans. The composition may additionally comprise antigenic materialas described herein and/or chitosan modified by an organic carboxylicacid, or a salt thereof.

In a further preferred embodiment the composition for use of the presentinvention comprises homogenised inactivated dermatophyte microconidia ofTrichophyton mentagrophytes, in particular Trichophyton mentagrophytesDSM-7279. The composition may additionally comprise antigenic materialas described herein and/or chitosan modified by an organic carboxylicacid, or a salt thereof.

In a further preferred embodiment the composition for use of the presentinvention comprises homogenised inactivated dermatophyte microconidia ofTrichophyton verrucosum, in particular Trichophyton verrucosumDSM-28406. The composition may additionally comprise antigenic materialas described herein and/or chitosan modified by an organic carboxylicacid, or a salt thereof.

In a further preferred embodiment the composition for use of the presentinvention comprises a mixture of homogenised inactivated dermatophytemicroconidia of Trichophyton verrucosum, in particular of Trichophytonverrucosum DSM-28406 and homogenized inactivated yeast blastospores ofCandida albicans, in particular of Candida albicans DSM-9456. Thecomposition may additionally comprise antigenic material as describedherein chitosan modified by an organic carboxylic acid, or a saltthereof.

In a further preferred embodiment the composition for use of the presentinvention comprises a mixture of homogenised inactivated dermatophytemicroconidia of Trichophyton mentagrophytes, Trichophyton verrucosum andTrichophyton sarkisovii. For example, a variation of the vaccinePolivac-TM comprising in comparision to the classical Polivac-TM noTrichophyton equinum and Microsporum strains (manufacturer: “Vetbiochim”LLC, Moscow; Distributor: “Prostore” LLC, Moscow) is in accordance withthis embodiment and can be used as a composition for use of the presentinvention. The composition may additionally comprise antigenic materialas described herein and/or chitosan modified by an organic carboxylicacid, or a salt thereof.

In a further preferred embodiment the composition for use of the presentinvention comprises a mixture of homogenised inactivated dermatophytemicroconidia of Trichophyton mentagrophytes, Microsporum canis,Microsporum canis var. obesum, Microsporum canis var distortum andMicrosporum gypseum. The composition may additionally comprise antigenicmaterial as described herein and/or chitosan modified by an organiccarboxylic acid, or a salt thereof.

In a further preferred embodiment the composition for use of the presentinvention comprises a mixture of homogenised inactivated dermatophytemicroconidia of Trichophyton mentagrophytes, Trichophyton verrucosum,Trichophyton equinum, Trichophyton sarkisovii, Microsporum canis,Microsporum canis var. obesum, Microsporum canis var distortum andMicrosporum gypseum. The composition may additionally comprise antigenicmaterial as described herein and/or chitosan modified by an organiccarboxylic acid, or a salt thereof.

In a further preferred embodiment the composition for use of the presentinvention comprises a mixture of homogenised inactivated dermatophytemicroconidia of Trichophyton mentagrophytes, Trichophyton verrucosum andTrichophyton sarkisovii. The composition may additionally compriseantigenic material as described herein and/or chitosan modified by anorganic carboxylic acid, or a salt thereof.

In a further preferred embodiment the composition for use of the presentinvention comprises homogenised inactivated yeast blastospores ofCandida albicans, in particular of Candida albicans DSM-9456. Thecomposition may additionally comprise antigenic material as describedherein and/or chitosan modified by an organic carboxylic acid, or a saltthereof.

In a further preferred embodiment the composition for use of the presentinvention comprises a mixture of homogenised inactivated dermatophytemicroconidia of Trichophyton mentagrophytes, Trichophyton verrucosum andTrichophyton sarkisovii. The composition may additionally compriseantigenic material as described herein and/or chitosan modified by anorganic carboxylic acid, or a salt thereof.

In a further preferred embodiment the composition for use of the presentinvention comprises antigenic material of yeast blastospores and/ordermatophyte microconidia. Especially preferred is a compositioncomprising antigenic material of dermatophyte microconidia ofTrichophyton verrucosum, in particular of the strain Trichophytonverrucosum DSM-28406.

In a further preferred embodiment the composition for use of the presentinvention comprises a mixture of homogenised inactivated dermatophytemicroconidia of one, two, three, four, five, six, seven, eight, nine orten of the above listed dermatophytes and antigenic material of one,two, three, four, five, six, seven, eight, nine or ten of the abovelisted dermatophytes and/or one, two, three or four of the above listedyeasts.

In a further preferred embodiment the composition for use of the presentinvention comprises a mixture of antigenic material of dermatophytemicroconidia of one, two, three, four, five, six, seven, eight, nine orten of the above listed dermatophytes with homogenized inactivated yeastblastospores of one, two, three or four Candida strains, in particularof Candida albicans, more particularly of Candida albicans DSM-9456,Candida albicans DSM-9457, Candida albicans DSM-9458, or Candidaalbicans DSM-9459.

In a further preferred embodiment the composition for use of the presentinvention comprises a mixture of antigenic material of blastospores ofone, two, three or four Candida strains, in particular of Candidaalbicans, more particularly of Candida albicans DSM-9456, Candidaalbicans DSM-9457, Candida albicans DSM-9458, or Candida albicansDSM-9459, with homogenised inactivated dermatophyte microconidia of one,two, three, four, five, six, seven, eight, nine or ten of the abovelisted dermatophytes.

The antigenic material of yeast blastospores and/or dermatophytemicroconidia comprises preferably polysaccharides and/or glycopeptidesisolated from keratinophilic fungi or yeasts. The antigenic materialcomprising such polysaccharide and/or glycopeptides can be antigenicnonsoluble material (ANMD), antigenic soluble material (ASMP) orantigenic exogenous material (AEMP). The keratinophilic fungi arepreferably of the species Trichophyton or Microsporum, more preferablyTrichophyton verrucosum, Trichophyton mentagrophytes, Trichophytonequinum, Trichophyton sarkisovii, Trichophyton rubrum, Trichophytonmentagrophytes, Microsporum gypseum and Microsporum canis, and thekeratinophilic yeasts are preferably of the species Candida, morepreferably Candida albicans. Especially preferred is antigenic materialderived from Trichophyton mentagrophytes DSM-7279, Trichophytonverrucosum DSM-28406, Trichophyton rubrum DSM-9469, Trichophyton rubrumDSM-9470, Trichophyton rubrum DSM-9471, Trichophyton rubrum DSM-9472,Candida albicans DSM-9456, Candida albicans DSM-9457, Candida albicansDSM-9458, and Candida albicans DSM-9459. The antigenic material is, forexample, obtainable by the method disclosed in WO 97/07232.

In general for obtaining ANMD, the fungal cells belonging to the groupof keratinophilic fungi or yeasts are treated under aqueous alkalineconditions, the solid and liquid phases of the preparation areseparated, and after separation the solid phase is treated with mineralor organic acid. The treatment under aqueous alkaline conditions ispreferably performed with about 0.1 to 5% (w/v) KOH or NaOH at about 20°C. to 150° C. for up to 30 h. The solid phase is preferably treated with0.2 to 1.5 M organic acid or 0.05 to 1 M mineral acid and washed with anaqueous solution. More specifically, the keratinophilic fungi or yeastsare preferably cultivated on Agar plates. One preferred medium is forexample malt extract agar from Oxoid. Other media that will ensuregrowth of keratinophilic fungi or yeast may be used as well. Theresulting fungal biomass is lifted off and treated with the aqueoussolution of alkali. Subsequently, the solid and liquid phases of thepreparation are separated, for example by centrifugation, filtration orsedimentation. Preferably, the separation is performed bycentrifugation, e.g. at 3500 g, which allows good separation of thefungal cell debris. Both the treatment under aqueous alkaline conditionsand the separation step may be repeated several times. After alkalinetreatment, the resulting supernatant is treated under the acidic aqueousconditions as outlined above. For example, HCl or acetic acid can beused. The treatment with acid is preferably performed for about 0.5 toabout 3 hours. The temperature is preferably in the range of about 70 toabout 100° C. The aqueous solution for washing is preferably distilledwater. Advantageously, the washing is repeated about five times.Finally, the solid phase is lifted off and homogenized in water forinjection or in an aqueous solution of 0.1-0.9% solution of chitosanmodified by an organic carboxylic acid, or a salt thereof. Thehomogenization is preferably performed in a volume of about 100 to about500 ml. The concentration of particles is then preferably adjusted toabout 30 to 90 million particles per ml. Finally, the preparationcomprising the antigenic material can be lyophilised and stored underdry conditions.

ASMP can generally be obtained as follows: Fungal cells ofkeratinophilic fungi or yeasts are treated under aqueous alkalineconditions, the solid and liquid phases of the preparation areseparated, after separation the supernatant is treated with mineral ororganic acid, and after separation ASMP is precipitated from thesupernatant. More particularly, keratinophilic fungi or yeasts arecultivated on Agar plates, for example as described in EP 0564620. Onepreferred medium is for example malt extract agar from Oxoid. Othermedia that will ensure growth of keratinophilic fungi or yeast may beused as well. The resulting fungal biomass is lifted off and treatedwith an aqueous solution of alkali. Preferred aqueous alkaline solutionsare NaOH or KOH at preferred concentrations of 0.1-5% (w/v). Alkalinetreatment is preferably performed at about 20-150° C. for up to 30 h.Following the processing under aqueous alkaline conditions, the solidand liquid phases of the preparation are separated, for example, bycentrifugation, filtration or sedimentation. Preferably, the separationis achieved by centrifugation, which ensures good separation of thefungal cell debris, for example, at forces of about 3500 g. Thetreatment under aqueous alkaline conditions, as well as the separationstep, may be repeated several times. After the alkaline treatment andseparation, the resulting supernatant is treated under acidic aqueousconditions, e.g. 0.2-1.5M organic acid or 0.05-1M mineral acid. Forexample, HCl or acetic acid can be used, preferably at pH values ofabout pH 2.5 to pH 4.5. Preferably, the treatment under aqueous acidicconditions is for about 2 to 4 hours at temperatures of about 4 to 8°C., whereafter separation of the solid and liquid layers takes place.The treatment under aqueous acidic conditions, as well as the separationstep, may be repeated several times, preferably under conditions asabove indicated. Then, the supernatant from the separation step issubject to a precipitation step. Preferably, the precipitation isperformed by adding a suitable organic solvent, e.g. an alcohol such asa lower alkanol, for example methanol or ethanol. A ratio of one volumesupernatant to 2-5 volumes of alcohol will result in a goodprecipitation of the antigenic material. Other nonalcoholicprecipitation procedures known to the person skilled in the art may beused as well, for example, ammonium sulphate or other saltprecipitation. The solid phase is then subject to a further separationstep, preferably under conditions as described above. The resultingsolid phase is recovered and, if desired, dissolved in an aqueoussolution, preferably in distilled water, typically in a volume of about25 to 100 ml. Finally, the ASMP preparation can be lyophilised andstored for prolonged time periods under dry conditions.

AEMP can generally be obtained as follows: fungal cells ofkeratinophilic fungi or yeasts are cultivated in liquid medium, thesolid phase and liquid phases of the preparation are separated, andafter separation AEMP is precipitated from the supernatant. Moreparticularly, keratinophilic fungi or yeasts may be incubated in aqueoussolution or cultivated in liquid medium. The cultivation may be for upto about 240 to 250 hours. The volume of the solution or culture is heredefined as primary volume (PV). Distilled water can be used as well asmedia described in EP 0564620. After incubation or cultivation, thefungal cells are separated, for example, by centrifugation, filtrationor sedimentation, preferably by centrifugation under conditions asdescribed above. Optionally, the resulting supernatant is thenlyophilised and subsequently dissolved in aequeous solution, preferablyin water. Preferably, the volume of water is about 0.1 to 0.2 volumes ofthe primary volume (PV). The resulting solution or the resultingsupernatant obtained after seperation is then subject to a precipitationstep. Preferably, the precipitation is performed by adding a suitableorganic solvent, e.g. an alcohol such as a lower alkanol, for examplemethanol or ethanol. A ratio of one volume supernatant to about 1 to 5volumes of alcohol will result in a good precipitation of the antigenicmaterial. Other nonalcoholic precipitation procedures known to theperson skilled in the art may be used as well, for example ammoniumsulphate or other salt precipitation. The resulting precipitate isrecovered and, if desired, dissolved in an aqueous solvent, preferablyin distilled water. Preferably, about 0.5 to 50 mg of the precipitateare dissolved in 1 ml aqueous solvent. Finally, the AEMP solution can belyophilised and stored for prolonged time periods under dry conditions,preferably at about 2 to 10° C.

In a highly preferred embodiment of the present invention thecomposition for use of the present invention comprises the strainTrichophyton verrucosum BINO 348-DSM 28406, antigenic material thereofand/or homogenised inactivated dermatophyte microconidia thereof. Thecomposition may additionally comprise antigenic material as describedherein and/or chitosan modified by an organic carboxylic acid, or a saltthereof or a hydro colloid according to the present invention.

In further highly preferred embodiments of the present invention thedermatophyte microconidia of Trichophyton verrucosum of the embodimentsas outlined above are dermatophyte microconidia of the strainTrichophyton verrucosum BINO 348-DSM 28406. However, the embodiments ofthe present invention as outlined above may also additionally comprisedermatophyte microconidia of the strain Trichophyton verrucosum BINO348-DSM 28406.

In a further highly preferred embodiment the composition for use of thepresent invention comprises a mixture of homogenised inactivateddermatophyte microconidia of Trichophyton verrucosum BINO 348-DSM 28406and homogenized inactivated yeast blastospores of Candida albicans. Thecomposition may additionally comprise antigenic material as describedherein and/or chitosan modified by an organic carboxylic acid, or a saltthereof a hydro colloid according to the present invention.

Thus, the present invention also refers to strain Trichophytonverrucosum DSM-28406. In a further aspect the present invention relatesto homogenised inactivated dermatophyte microconidia of Trichophytonverrucosum DSM-28406. Further, the present invention relates toantigenic material, including ANMP, AEMP and ASMP, of dermatophytemicroconidia of Trichophyton verrucosum DSM-28406.

In addition, the present invention refers to the strain Trichophytonverrucosum DSM-28406, homogenised inactivated dermatophyte microconidiaof Trichophyton verrucosum DSM-28406 and antigenic material, includingANMP, AEMP and ASMP, of dermatophyte microconidia of Trichophytonverrucosum DSM-28406 for use in human and/or veterinary medicine. Inparticular, the present invention refers to the strain Trichophytonverrucosum DSM-28406 homogenised inactivated dermatophyte microconidiaof Trichophyton verrucosum DSM-28406 and antigenic material ofdermatophyte microconidia of Trichophyton verrucosum DSM-28406 for usein a method of treating and/or preventing of hoof- and claw diseases inanimals, lameness, digital dermatitis, interdigital dermatitis,interdigital phlegmone and dermatophytosis in animals and warts inhumans. The animals are preferably mammals, more preferably bovidaeand/or pigs, most preferably cattle.

In addition, the present invention refers to a composition comprisinghomogenised inactivated dermatophyte microconidia of the strainTrichophyton verrucosum DSM-28406. The present invention also refers toa composition comprising antigenic material of dermatophyte microconidiaof the strain Trichophyton verrucosum DSM-28406. The compositions of thepresent invention are preferably pharmaceutical compositions. Thepresent invention also refers to a composition comprising homogenisedinactivated dermatophyte microconidia of the strain Trichophytonverrucosum DSM-28406 for use in human and/or veterinary medicine, inparticular for use in a method of treating and/or preventing hoof- andclaw diseases in animals and lameness, digital dermatitis, interdigitaldermatitis, interdigital phlegmone, dermatophytosis in animals and wartsin humans. The present invention also refers to a composition comprisingantigenic material of dermatophyte microconidia of the strainTrichophyton verrucosum DSM-28406 for use in human and/or veterinarymedicine, in particular for use in a method of treating and/orpreventing hoof- and claw diseases in animals and lameness, digitaldermatitis, interdigital dermatitis, interdigital phlegmone,dermatophytosis in animals and warts in humans. The animals arepreferably mammals, more preferably bovidae and/or pigs, most preferablycattle.

Antigenic material, including ANMP, ASMP and AEMP, of dermatophytemicroconidia of the strain Trichophyton verrucosum DSM-28406 isobtainable by the same method as outlined above for antigenic materialof yeast blastospores and/or dermatophyte microconidia in general.

The composition for use of the present invention and the composition ofthe present invention are summarized in the following as “compositionsof the present invention”. Compositions of the present inventioncomprising dermatophyte microconidia of only one strain or yeastblastospores of only one strain can be prepared as follows:

-   (a) growing a dermatophyte and a yeast, respectively, on suitable    solid medium, harvesting and homogenising the dermatophyte, and-   (b) inactivating the homogenate obtained in step (a)

The compositions of the present invention comprising a mixture ofdermatophyte microconidia and/or yeast blastospores can be prepared asfollows:

-   (a) growing one dermatophyte strain and two, three, four, five, six,    seven, eight, nine or ten distinct strains of dermatophytes,    respectively, separately on suitable solid medium, harvesting each    culture and homogenising each culture separately, and-   (b) optionally, growing one yeast strain and two, three or four    distinct strains of yeast, respectively, separately on suitable    solid medium, harvesting each culture and homogenising each culture    separately, and-   (c) combining and inactivating the homogenates obtained in step (a)    and optionally obtained in step (b).

The growing of the dermatophytes of the above described preparationprocesses is preferably done on agar and wort in culture flasks.Preferably, the culture is performed for about 15 to about 30 days.Preferably, the cultivation is performed at a temperature of about 26°C. to about 28° C.

The growing of the yeasts of the above described preparation processesis preferably done on malt extract-agar or agar Sabouraud in cultureflasks. Preferably, the culture is performed for about 4 to about 7days. Preferably, the cultivation is performed at a temperature of about28 to about 37° C.

After cultivation the dermatophytes and yeasts, respectively, step ishomogenized to obtain a fine suspension. Preferably the homogenizationis performed in deionized water, in an aqueous solution comprising about0.1 to 0.3% fermented hydrolyzed muscle protein or about 0.1 to 1% soyor pork peptone in combination with about 5 to 6% glucose and about 0.1to 1% yeast extract, or in an aqueous solution comprising 0.1-0.9% (w/v)chitosan modified by an organic carboxylic acid, or a salt thereof.

Suitable volumes for homogenization are about 100 to 500 ml. Preferably,the concentration of microconidia and blastospores, respectively, isadjusted to about 30 to about 90 million microconidia and blastospores,respectively, per ml or to about 250 to about 500 thousand, morepreferably about 250 to about 400 thousand microconidia andblastospores, respectively, per ml. Then, the suspension may optionallybe additional adjusted to about 40, 50 or 60 million of microconidia andblastopores, respectively, per ml or to about 250 to about 500 thousand,more preferably to about 250 to about 400 thousand microconidia andblastospores, respectively, per ml with distilled water, physiologicalsalt solution as e.g. sodium chloride or another suitable solution.

In case of the preparation of a mixture, the single suspensions arepreferably adjusted to the same amount of microconidia and blastospore,respectively, per ml and equal volumes of each culture in suspension aremixed in a single container.

The inactivation is preferably performed by using thiomersal,formaldehyde and/or 2-propiolactone. The agents for inactivating can beadded directly to the cell suspension. Preferred is an inactivation byadding thiomersal in a ratio of about 1:11000 to about 1:25000 (w/v).Also preferred is an inactivation by adding formaldehyde to reach an endconcentration of about 0.2% to about 0.4% (v/v). Subsequently, themixture is preferably incubated. The incubation can be performed forabout 1 to 30 days at a temperature of about 20° C. to about 37° C.Preferred is incubation for about 1 to 3 days at room temperature, forabout 5 to 7 days at 37° C., for about 30 days at room temperature orfor about 30 days at about 26° C. to 28° C.

In a preferred embodiment the microconidia of the compositions of thepresent invention are in a swollen condition and/or have germ tubes.More preferably, at least 50% of the blastospores and/or microconidiaare in a swollen condition and/or have germ tubes.

The swollen condition and/or the germ tubes of dermatophytes can e.g. beobtained by a second incubation step. Said second incubation step ispreferably performed after the homogenization and before inactivation asdescribed above. For performing the second cultivation step themicroconidia suspension is placed in a separate vessel containing thesame medium of the first incubation step. The second cultivation step ispreferably performed for about 10 to about 48 hours. The secondcultivation step is preferably performed at a temperature of about 28°C. Preferably, the second cultivation step is continued until at least50% of the microconidia display a swollen or germinating condition andno more than about 7 to 10% of the cells display a second mycelialbranch. The diameter of swollen and germinated microconidia is increasedby about 1.2 or more compared to regular microconidia.

The chitosan modified by an organic carboxylic acid, or a salt thereofcan also be called chitosan variant or chitosan derivative. It ispreferably obtainable by contacting chitosan with an organic carboxylicacid or salt thereof. Said contact is preferably performed by incubatingchitosan in an aqueous solution of an organic carboxylic acid or a saltthereof, more preferably by incubating chitosan in an aqueous solutionof valeric acid, lactic acid, para-aminobenzoic acid or glucuronic acidor a salt thereof, in particular chloride of valeric acid. Preferably,said incubation is performed by mixing and/or under stirring.

Thus, the chitosan modified by an organic carboxylic acid or a saltthereof is preferably obtainable by a method comprising

-   (a) incubating chitosan in an aqueous solution of an organic    carboxylic acid or a salt thereof.

In a preferred embodiment the chitosan is firstly dissolved under acidicaqueous conditions and subsequently precipitated by increasing the pHvalue to a pH value of about 8.0 to about 8.5 before it is incubated inthe aqueous solution of the organic carboxylic acid or the salt thereofas described above.

Thus, the chitosan modified by an organic carboxylic acid, or a saltthereof is preferably obtainable by a method comprising dissolvingchitosan in an aqueous solution of an acid

-   -   (ii) increasing the pH value until chitosan is precipitated    -   (iii) recovering the precipitated chitosan, and

-   (a) incubating the recovered chitosan of step (iii) in an aqueous    solution of an organic carboxylic acid or a salt thereof.

The organic carboxylic acid or a salt thereof of step (a) has preferablya pKs of about 2 to about 5, more preferably of about 2.3 to about 4.9.More preferably said organic carboxylic acid is valeric acid, lacticacid, para-aminobenzoic acid or glucuronic acid or a salt thereof, inparticular chloride of valeric acid. Said carboxylic organic acid or asalt thereof is preferably used in a concentration of about 0.2 M toabout 22.5 M. The incubation of chitosan and the recovered chitosan ofstep (iii), respectively, and the aqueous solution of the organiccarboxylic acid or a salt thereof as outlined in step (a) results in thesolution of the chitosan, the modification of the chitosan and/or theformation of a gel. Preferably, the pH value of the aqueous solution instep (a) is about 5 to about 6 or about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5,5.6, 5.7, 5.8, 5.9 or 6.0. Preferably, the modification of the chitosantakes place in an aqueous solution comprising about 1 mM to about 100 mMof the organic carboxylic acid or a salt thereof, more preferably about1 mM to about 10 mM.

Preferably, step (a) is performed until the chitosan is modified anddissolved. It is preferably performed by mixing chitosan with an aqueoussolution of the organic carboxylic acid or a salt thereof or bysuspending chitosan under aqueous conditions and adding the organiccarboxylic acid to the suspension. It is preferably performed understirring for about 1 to about 72 hours, more preferably for about 24 toabout 48 hours. Step (a) may comprise the addition of a further acid ormay be performed in the presence of a further acid. Said further acid ispreferably a mineral acid, an organic acid or a salt of said mineralacid or organic acid. Preferably, the mineral acid is HCl or H₂SO₄ andthe organic acid is glutamic acid, para-aminobenzoic acid or lacticacid. The mineral or organic acid is preferably added or present in anamount to adjust the pH value of the mixture of step (a) to a pH valueof about 5 to about 6 or about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9 or 6.0. The addition of a further acid may support thedissolution and/or the modification of the chitosan e.g. by decreasingthe time which is necessary to dissolve the modified chitosan.

The concentration of chitosan in step (a), or if the method comprises astep (i) for step (i), is preferably about 1 g to about 20 g chitosanper liter, more preferably about 5 g to about 15 g chitosan per liter,most preferably about 8 to about 10 g chitosan per liter.

The chitosan used for step (a), or if the method comprises a step (i)for step (i), may be commercially available chitosan or chitosanisolated from any natural source comprising chitosan such as biomasscomprising chitosan. Alternatively, chitin may be used which isdeacetylated to obtain chitosan prior to step (a), or if the methodcomprises a step (i) prior to step (i). Said chitin may be commerciallyavailable or it may be isolated from a natural source comprising chitinsuch as biomass comprising chitin. The biomass for chitin and/orchitosan isolation is preferably biomass of fungi, insects and/orcrustaceans. The deacetylation of chitin can be performed by knownmethods in the art as e.g. by using sodium hydroxide (NaOH) in excess asa reagent and water as a solvent or by enzymatic methods. The isolationof chitosan and/or chitin from natural sources can also be performed byknown methods in the art.

The chitosan used for step (a), or if the method comprises a step (i)for step (i), has preferably a degree of deacetylation of about 62% toabout 95%, more preferably of about 80% to about 94%, more preferably ofabout 89% to about 93% or of about 93% to about 98%, of about 93% to95%, of about 95% to about 98%, or of at least 60%, more preferably ofat least 70%, 80%, 90% or 95%, or a degree of deacetylation of about 60%to about 100%, more preferably of about 80% to about 95%, even morepreferably of about 90% to about 95%, most preferably about 77% to about80%. The chitosan used for step (a), or if the method comprises a step(i) for step (i), has preferably a molecular weight or an averagemolecular weight of about 50 Da to about 700 kDa, in particular of about15 kDa to about 500 kDa, more particular of about 15 kDa to about 150kDa, or of about 80 kDa to about 200 kDa, of about 150 kDa to about 300kDa, of about 100 kDa to about 250 kDa or of about 300 kDa to about 700kDa.

The chitosan used for step (a), or if the method comprises a step (i)for step (i), has preferably a viscosity of about 50 to 400 MPas, morepreferably about 70 to about 150 MPas or about 151 to about 350 MPas.

Before the chitosan is used in step (a), or if the method comprises astep (i) in step (i), the chitosan may be sterilized by autoclaving.Said sterilization may result in that the modified chitosan is lesstoxic, better tolerated by any subject and/or results in less unintendedside-effects.

Preferably, step (i) is performed by the use of an aqueous solution ofan weak acid, preferably by an organic acid or a salt thereof, morepreferably by acetic acid, valeric acid, lactic acid, para-aminobenzoicacid or glucuronic acid or a salt thereof, in particular chloride ofvaleric acid. The acid is preferably used in a concentration of about0.8% to about 2%. Step (i) is preferably performed under stirring. Thestirring may be performed for about 2 hour to about 24 hours.Preferably, step (i) is performed until a gel or gel suspension isobtained. Unsolved particles may be removed, e.g. by filtration. Forexample, a metal grid with a cell of 200 μm to 300 μm may be used forsuch a filtration.

Step (ii) is preferably performed by increasing the pH value of the gelor gel suspension obtained in step (i) until a precipitate is formed. Itis preferably performed under stirring. It is preferably performed bytreating chitosan under aqueous alkaline conditions, more preferablyunder aqueous alkaline conditions comprising about 0.1 to about 25.0%alkali. In a preferred embodiment the alkali is NaOH. Preferably, saidstep is performed at a temperature of about 4° C. to about 55° C.Preferably, the treatment is performed for about 20 min to about 2hours, more preferably for about 30 min to about 70 min, but it may alsotake up to about 24 h. Preferably, the pH value is increased by addingthe alkali to the gel or gel suspension of step (i). Preferably, the pHvalue is increased to obtain a pH of about 8.0 to about 8.5. Step (ii)may result in a further deacetylation of the chitosan. It may alsoresult in that the modified chitosan is less toxic, better tolerated byany subject and/or results in less unintended side-effects.

Step (iii) is preferably performed by centrifuging the mixture orsuspension obtained in step (ii). The centrifugation is preferablyperformed at about 4000 to about 6000 revolution/min, more preferably atabout 5000 revolution/min. The centrifugation is preferably performedfor up to 60 minutes.

The methods by which the modified chitosan is obtainable may compriseadditional steps. For example, the product obtained in step (ii), may behomogenized. Preferably, the step of homogenization is performed in aclosed sterile homogenizer. Alternatively or in addition, the productobtained in step (a) may be dialyzed. The dialysis is preferablyperformed in a closed system to remove free ions of salts and lowmolecular weight compounds. Preferably, the dialysis is performed bycross filtration for about 1 to about 6 hours or by membrane filtrationagainst distillate water for about 24 to about 48 hours.

Alternatively or in addition, the methods by which the modified chitosanis obtainable may comprise a further step of preparing the finalproduct. The preparation of the final product may comprise the dilutionof the obtained product. Preferably, the product is diluted by theaddition of water, more preferably of sterile water for injection.However, the product may also be diluted in any other suitable aqueoussolution. Alternatively or in addition, the preparation of the finalproduct may comprise the addition of one or more further compounds, suchas diluents or preservatives. Suitable preservatives are for examplechlorocresol, thiomersal and formalin. Finally, the final product may besterilized. Preferably the sterilization is performed by heating,preferably for about 40 to 50 minutes at a temperature of about 65° C.to about 80° C. Preferably, said sterilization is repeated one, two,three, four or five times.

Preferably, the final product has a concentration of about 0.02 g toabout 2 g modified chitosan per liter, more preferably of about 0.04 toabout 1 g modified chitosan per liter.

In a preferred embodiment the methods by which the modified chitosan isobtainable comprise the steps as described in the Examples. For example,the methods may comprise the following steps:

-   -   optionally sterilizing chitosan e.g. by autoclaving,

-   (i) dissolving chitosan in an aqueous solution of an acid, in    particular in the presence of an acetic acid,    -   optionally removing undissolved particles e.g. by filtration,

-   (ii) increasing the pH value until chitosan is precipitated,

-   (iii) recovering the precipitated chitosan,    -   optionally homogenizing the recovered chitosan under aqueous        conditions,

-   (a) incubating the recovered chitosan of step (iii) or the    homogenized recovered chitosan in an aqueous solution of an organic    carboxylic acid or a salt thereof, optionally in the presence of a    further mineral acid or organic acid,    -   optionally dialyzing the product obtained in step (a),    -   optionally adding further compounds, such as diluents or        preservatives, and    -   optionally sterilizing the final product e.g. by heating.

Preferably, the order of the steps as outlined above corresponds to theorder as listed above. However, as known by the person skilled in theart the order of single steps may be varied as long as the same effectsare achieved. For example, diluents such as water may be added invarious stages of the method as described above.

In a preferred embodiment of the present application the modifiedchitosan, chitosan derivative or chitosan variant is a Polyamino-sugarcolloid, preferably a hydro colloid. In another preferred embodiment ofthe present application the hydro colloid is a Chitosan-Glucuronicacid-Hydro-Colloid or Chitosan-p-Aminobenzoic acid-Hydro-Colloid orChitosan-Valeric acid-Hydro-Colloid. In another preferred embodiment ofthe present application the Chitosan-Glucuronic acid-Hydro-Colloid hasthe chemical formula: (C₆H₁₁O₄N)_(x) (C₈H₁₃O₅N_(y) (C₆H₁₀O₇)_(z)(H₂O)_(m). Preferably, the Chitosan-Glucuronic acid-Hydro-Colloid hasthe following molecular weight: x*(161)+y*(203)+z*(194.14)+m*(18). Inanother preferred embodiment of the present application theChitosan-p-Aminobenzoic acid-Hydro-Colloid has the chemical formula:(C₆H₁₁O₄N)_(x) (C₈H₁₃O₅N)_(y) (C₇H₇O₂N)_(z) (H₂O)_(m). Preferably, theChitosan-p-Aminobenzoic acid-Hydro-Colloid has the following molecularweight: x*(161)+y*(203)+z*(137.14)+m*(18). In another preferredembodiment of the present application the Chitosan-Valericacid-Hydro-Colloid has the chemical formula: (C₆H₁₁O₄N)_(x)(C₈H₁₃O₅N)_(y) (C₆H₁₀O₇)_(z) (HCl)_(z) (H₂O)_(m). Preferably,Chitosan-Valeric acid-Hydro-Colloid has the following molecular weight:x*(161)+y*(203)+z*(102)+z*(36.5)+m*(18).

In another preferred embodiment of the present application the modifiedchitosan, chitosan derivative or chitosan variant is a natural white toyellowish viscous liquid. Preferably, the modified chitosan, chitosanderivative or chitosan variant has typical odor of the carboxylic acid,preferably the typical odor of valeric acid.

In another preferred embodiment, the modified chitosan, chitosanderivative or chitosan variant contains about 0.2% pentanoly chloride,or 0.2% Glucuronic acid, or 0.2% p-Aminobenzoic acid.

In another preferred embodiment, the modified chitosan, chitosanderivative or chitosan variant contains about 1.0% pentanoly chloride.

In another preferred embodiment, the modified chitosan, chitosanderivative or chitosan variant contains 1% chitosan residue from dryingchitosans.

In another preferred embodiment, the modified chitosan, chitosanderivative or chitosan variant has about 10 to about 1000 mOsmol,preferably about 10 to about 200 mOsmol, most preferably about 100mOsmol.

In another preferred embodiment, the modified chitosan, chitosanderivative or chitosan variant is a hydro colloid comprising:

-   (i) 0.1% to 5% (w/v) chitosan and 0.001 to 5% (w/v) valeric acid, or    a salt thereof, preferably chloride of valeric acid or-   (ii) 0.1% to 5% (w/w) chitosan and 0.001 to 5% (w/w) glucuronic acid    or p-aminobenzoic acid or a salt thereof.

In another preferred embodiment, the modified chitosan, chitosanderivative or chitosan variant is a hydro colloid comprising:

-   (i) 0.1% to 3% (w/v) chitosan and 0.001 to 2% (w/v) valeric acid or    a salt thereof, preferably chloride of valeric acid, or-   (ii) 0.1% to 3% (w/w) chitosan and 0.001 to 2% (w/w) glucuronic acid    or p-aminobenzoic acid or a salt thereof.

In another preferred embodiment, the modified chitosan, chitosanderivative or chitosan variant is a hydro colloid comprising:

-   (i) 0.1% to 1.2% (w/v) chitosan and 0.001 to 1% (w/v) valeric acid    or a salt thereof, preferably chloride of valeric acid, or-   (ii) 0.1 to 1.2% (w/w) chitosan and 0.001 to 1% (w/w) glucuronic    acid or p-aminobenzoic acid or a salt thereof.

In another preferred embodiment, the modified chitosan, chitosanderivative or chitosan variant is a hydro colloid comprising:

-   (i) 0.1% to 1.2% (w/v) chitosan and-   (ii) 0.01 to 0.44% (w/v) valeric acid, or a salt thereof, preferably    chloride of valeric acid.

In another preferred embodiment, the modified chitosan, chitosanderivative or chitosan variant is a hydro colloid comprising:

-   (i) 0.1% to 1.2% (w/w) chitosan and-   (ii) 0.001 to 0.6% (w/w) glucuronic acid or a salt thereof.

In another preferred embodiment, the modified chitosan, chitosanderivative or chitosan variant is a hydro colloid comprising:

-   (i) 0.1% to 1.2% chitosan and-   (ii) 0.006 to 1% (w/w) p-aminobenzoic acid or a salt thereof.

Preferably the chitosan of the hydro colloid is a compound of formula[X]n, in which n represents an integer of about 1 to about 5000, inparticular an integer of about 300 to about 4000, and X has thefollowing formula (1):

wherein about 2% to about 38%, more preferably about 5% to about 20% ofthe X residues constituting said compound are modified by acetylationand wherein all or part of the X residues constituting said compound aremodified by an organic carboxylic acid or a salt thereof,

In a preferred embodiment, the remaining percentage of the hydro colloidaccording to the present invention is provided by the dispersion media,preferably water or water and hydrogenchloride (HCl).

In a preferred embodiment, the hydro colloid according to the presentinvention is used as a dilution, preferably in a dilution of 0 to 10times.

In an exemplified embodiment the modified chitosan is obtainable by thefollowing procedure:

Chitosan with deacetylation of e.g. 67%, a viscosity of 50-200 mPas canbe used as raw material. 40 grams of chitosan is sterilized byautoclaving and 3.5 liters of water for injection are added understirring. 40 ml of acetic acid are added to the obtained suspension. Thefinal volume is adjusted to a volume of 4 liters with water forinjection. The mixture is stirred in a sterile container for about 24hours until a gel suspension is obtained. Unsolved particles are removedby filtration through a metal grid with a cell size of 200 μm-300 μm. 4N sodium hydroxide (NaOH) in added dropwise to the prepared mixture toobtain a final pH of 8.0. Upon that white flakes precipitate whichcontain the chitosan. The suspension is stirred for about 30 minutes.Under constant stirring, 4 mL valerian acid chloride is added dropwiseto the suspension. The obtained suspended material is stirred for onehour. Flakes and unsolved particles are separated and subsequentlyresuspended in 4 liters of sterile water for injections. Under stirring4N hydrochloric acid is added to obtain a pH of 5.0. The resultant gelis dialysed in a closed system to remove free ions of salts and lowmolecular weight compounds. After dialysis, the final product isprepared. For obtaining the final product 3 liters of the modifiedchitosan gel are adjusted to a volume of 25 liters by adding sterilewater for injection under stirring. Then, 500 ml of chlorocresolsolution containing 30 grams of chlorosresol are added to the mixture.The resultant suspension is adjusted to a volume of 30 liters. Theresultant sterile product is dispensed into vials under sterileconditions.

If no specific temperature ranges are given for the method steps asdescribed herein, the steps are preferably performed at room temperatureand/or in a range of about 10° C. to about 40° C., more preferably in arange of about 20° C. to about 30° C.

In a preferred embodiment the compositions of the present invention havea concentration of about 40 to about 90 million blastospores per ml,highly preferred is a concentration of about 40 million to 60 millionspores per ml. These concentrations are especially preferred if thecompositions are for intramuscular administration. In a furtherpreferred embodiment the compositions of the present invention have aconcentration of about 0.2 to about 0.4 million spores per ml, highlypreferred is a concentration of about 0.25 million to about 0.3 millionspores per ml. These concentrations are especially preferred if thecompositions are for intracutaneous intramuscular administration.

In a further preferred embodiment the compositions of the presentinvention have a concentration of about 40 to about 60 million particlesof antigenic material of dermatophyte microconidia and/or yeastblastospores per ml.

In a further preferred embodiment the concentration of the chitosanmodified by an organic carboxylic acid, or a salt thereof is preferablyin the range of about 0.1% to about 0.9% (w/v), more preferably in therange of about 0.1 to about 0.3% (w/v), more preferably in the range ofabout 0.1 to about 0.3% (w/v).

Surprisingly, it was found that if the composition of the presentinvention additionally comprise chitosan modified by an organiccarboxylic acid, or a salt thereof the homogenised inactivateddermatophyte microconidia and/or inactivated homogenized yeastblastospores can be used in a about 50 times less dose.

The compositions of the present invention may additionally comprisepharmaceutically acceptable carrier, excipients and/or supports.

The compositions of the present invention are able to modulate theimmune system, i.e. they have immunostimulatory properties. They can beused as a vaccine for preventing the subject from the diseases asoutlined above. Alternatively or in addition, they can be used to treatand cure the subject from the diseases as outlined above. Thecompositions can be administered by known administration routes as e.g.parenterally, by intramuscular injection, by intracutaneous injection,by percutaneous injection and/or topically, preferably cutaneously. Theymay be administered in the absence or in the presence of one or moreadditional immunostimmulatory substances. In one embodiment said one ormore additional immunostimulatory substances are administered separatelyto the compositions of the present invention. In another embodiment theone or more additional immunostimulatory substances are comprised in oradded to the compositions of the present invention.

Said one or more immunostimulatory substance is preferably an adjuvant,preferably selected from the group consisting of vitamin-E acetate,o/w-emulsion, aluminum phosphate, aluminum oxide, aluminumhydroxide/methyl cellulose gel, an oil-emulsion, muramil-dipeptides,Freund's adjuvants and saponins and/or at least one cytokine, preferablyselected from the group consisting of IL 2, IL 12 and INF-Gamma.

In a more preferred embodiment the compositions of the present inventionadditionally comprise chitosan modified by an organic carboxylic acid,or a salt thereof or a hydro colloid according to the present invention.

In a preferred embodiment the compositions of the present invention is avaccine and/or is used as a vaccine.

The present invention relates also to a pharmaceutical compositioncomprising antigenic material of keratinophilic fungi or keratinophilicyeasts as described above. Such pharmaceutical composition mayadditionally comprise a pharmaceutical acceptable diluent, excipient orcarrier.

In a further aspect the present invention relates to a compositioncomprising antigenic material of keratinophilic fungi or keratinophilicyeasts as described above for use in a method of treatment of the animalbody by therapy. Such method typically comprises administering to asubject an effective amount of antigenic material of keratinophilicfungi or keratinophilic yeasts as described above or a composition or apharmaceutical composition as described above. The subject may forexample an animal, in particular a mammal, more preferably bovidaeand/or pigs, most preferably cattle. In particular, the antigenicmaterial of keratinophilic fungi or keratinophilic yeasts as describedabove or a composition or a pharmaceutical composition as describedabove may be used in methods for the treatment or prevention of hoof-and claw diseases as described above. The method of treatment maycomprise the treatment and/or prevention of bacterial, mycotic and/orviral infections of the skin, the leg, the hoof, the claws, the back ofthe foot and/or the interdigital space. Said infections may be caused byDichelobacter nodosus, Fusobacterium necroforun, Fusobacterium spp,Treponema spp such as T. phagedenis, T. vincentii, and T. denticola,Campylobacter spp, Staphylococcus aureus, Escherichia coli,Arcanobacterium pyogenes, and Prevotella spp. and/or a virus.

The dosage and route of administration used in a method of treatment(and/or prophylaxis) according to the present invention depends on thespecific disease/site of infection to be treated. The route ofadministration may be for example parenterally, by intramuscularinjection, by intracutaneous injection, by percutaneous injection,topically, cutaneously or any other route of administration. Forexample, the dosage and route of administration used in a method oftreating and/or preventing hoof- and claw diseases, in particular ID, DDand/or IP, in cattle may be as follows:

interval between concentration drug route of [million blastspores/frequency of number administration dose administration microconidia/ml]drug administration of sites [days] [ml] intramuscular 10-150, more 1,2, 3, 4 or 5 1 or 2 5-21 days, 0.5-10, preferably 30-90, more more morepreferably 40-60, or 20, preferably preferably 30, 40, 50, 60, 70, 80,7-10 days 1-5, or 0.5, 90, or 100 or 5, 6, 7, 1, 2, 2.5, 3, 8, 9, 10, 4,5, or 6 11, 12, 13 or 14 days intracutanous 0.1-0.6, more 1, 2, 3, 4 or5 1, 2 or 3 5-21 days, 0.2-0.6, preferably 0.25-0.4, or more more 0.1,0.2, 0.25, 0.3, 0.4, preferably preferably 0.5, or 0.6 7-10 days,0.3-0.5, or or 5, 6, 7, 0.2, 0.3, 8, 9, 10, 0.4. 0.5, or 11, 12, 13, 0.614, 15, 16, 17, 18, 19, 20 or 21 days

The following examples explain the present invention but are notconsidered to be limiting.

EXAMPLE 1

Vaccine Polivac-TM (manufacturer: “Vetbiochim” LLC, Moscow; Distributor:“Prostore” LLC, Moscow) against dermatopytosis of animals was used forthe prophylaxis and treatment of digital and/or interdigital dermatitisand/or interdigital phlegmon in cattle as described below.

EXAMPLE 2

Vaccine Polivac-T (manufacturer: “Vetbiochim” LLC, Moscow; Distributor:“Prostore” LLC, Moscow) against dermatopytosis of cattle was used forthe prophylaxis and treatment of interdigital and/or digital dermatitisand/or interdigital phlegmon in cattle as described below.

EXAMPLE 3

Dermatophyte culture of the species Trichophyton mentagrophytes DSM-7279is cultivated on agar/wort, for example in 4 Roux flasks. Each cultureis cultivated for 18 days at 28° C.

The fungal masses are lifted off and homogenised in 500 ml deionizedwater. Then suspension of microconidia is adjusted to 60 million ofmicroconidia per ml with physiological sodium chloride salt solution.The homogenate is inactivated by adding formaldehyde to 0.4% (v/v) inend directly to the cell suspension. The mixture is incubated for 5-7days at 37° C. The resulting composition is bottled, checked forsterility, safety and amount of microconidia in accordance with usualknown methods and can be stored refrigerated at 4-10° C. Vaccineobtainable according to this method was used for the prophylaxis andtreatment of interdigital digital dermatitis and/or interdigitalphlegmon in cattle as described below.

EXAMPLE 4

Dermatophyte culture of the species Trichophyton verrucosum DSM-28406 iscultivated on agar/wort, for example in 10 Roux flasks. Each culture iscultivated for 25 days at 26° C.

The fungal masses are lifted off and homogenised in 300 ml deionizedwater. The concentration of microconidia is adjusted to 80 million perml for each homogenate. Then suspension of microconidia is adjusted to40 million of microconidia per ml with distilled water. The homogenateis inactivated by adding formaldehyde to reach 0.5% (v/v) directly tothe cell suspension. The mixture is incubated for 5 days at 37° C. Theresulting composition is bottled, checked for sterility, safety andamount of microconidia in accordance with usual methods and stored at4-8° C. Vaccine obtainable according to this method was used for theprophylaxis and treatment of interdigital, digital dermatitis andinterdigital phlegmon in cattle as described below.

EXAMPLE 5

Dermatophyte culture of the species Trichophyton verrucosum DSM-28406 iscultivated on agar/wort, for example in 8 Roux flasks. Each culture iscultivated for 30 days at 27° C.

The fungal masses are lifted off and homogenised in 400 ml deionizedwater. The concentration of microconidia is adjusted to 70 million perml for each homogenate. Then suspension of microconidia is adjusted to60 million of microconidia per ml with distilled water. The homogenateis inactivated by adding formaldehyde to reach 0.3% (v/v) directly tothe cell suspension. The mixture is incubated for 7 days at 37° C. Theresulting composition is bottled, checked for sterility, safety andamount of microconidia in accordance with usual methods and stored at4-8° C. Vaccine obtainable according to this method was used for theprophylaxis and treatment of interdigital, digital dermatitis andinterdigital phlegmon in cattle as described below.

EXAMPLE 6

Dermatophyte culture of the species Trichophyton verrucosum DSM-28406 iscultivated on agar/wort, for example in 9 Roux flasks. Each culture iscultivated for 25 days at 28° C.

The fungal mass of the dermatophyte are lifted off and homogenised in500 ml an aqueous solution of 0.3% fermented hydrolyzed muscle proteinin combination with 5% glucose and 0.1% yeast extract. The concentrationof microconidia is adjusted to 40 million per ml for homogenate. Thenthe suspension of microconidia is fermented for 1 day at 28° C., until65% of the microconidia have germ tubes. After fermentation the cellsuspensions is washed with physiological solution of sodium chloride.The homogenate is inactivated by adding thiomersal in a ratio of 1:25000(w/v) directly to the cell suspension. The mixture is incubated for 30days at room temperature. The resulting composition is bottled, checkedfor sterility, safety and immunogenic properties in accordance withaccepted methods and can be stored refrigerated at 4-10° C. Compositionobtainable according to this method was used for the prophylaxis andtreatment of interdigital, digital dermatitis, interdigital phlegmon anddermatophytosis in cattle as described below.

EXAMPLE 7

A variation of the vaccine Polivac-TM against dermatopytosis of animalscomprising in comparision to the classical Polivac-TM no Trichophytonequinum and Microsporum strains (manufacturer: “Vetbiochim” LLC, Moscow;Distributor: “Prostore” LLC, Moscow) was used for the prophylaxis andtreatment of interdigital and/or digital dermatitis and/or interdigitalphlegmon in cattle as described below.

EXAMPLE 8

Vaccine Polivac-TM against dermatopytosis of animals (manufacturer:“Vetbiochim” LLC, Moscow; Distributor: “Prostore” LLC, Moscow) was usedfor the prophylaxis and treatment of interdigital and/or digitaldermatitis and/or interdigital phlegmon in cattle as described below.

EXAMPLE 9

Vaccine Polivac-TM against dermatopytosis of animals (manufacturer:“Vetbiochim” LLC, Moscow; Distributor: “Prostore” LLC, Moscow) was usedfor the prophylaxis and treatment of digital and/or interdigitaldermatitis and/or interdigital phlegmon in cattle as described below.

EXAMPLE 10

Vaccine Polivac-T against dermatopytosis of cattle (manufacturer:“Vetbiochim” LLC, Moscow; Distributor: “Prostore” LLC, Moscow) was usedfor the prophylaxis and treatment of interdigital and/or digitaldermatitis and/or interdigital phlegmon in cattle as described below.

EXAMPLE 11

Dermatophyte culture of the species Trichophyton verrucosum DSM-28406 iscultivated on agar/wort, for example in 10 Roux flasks. The culture iscultivated for 28 days at 28° C.

The fungal mass is lifted off and homogenised in 500 ml deionized water.The concentration of microconidia is adjusted to 50 million per ml inhomogenate. Then suspension of microconidia is adjusted to 50 million ofmicroconidia per ml with distilled water.

The species Candida albicans DSM-9456 is cultivated on malt extract-agaror agar Sabouraud, for example in 3 Roux flasks. Culture is cultivatedfor 4 days at 30° C. The blastospores are washed off with aphysiological solution of sodium chloride. The concentration ofblastospores in suspension is adjusted to 60 million per ml. Equalvolumes of each culture in suspension are mixed in a single container.The homogenates are inactivated by adding formaldehyde to reach 0.4%(v/v) in cell suspension. The mixture is incubated for 6 days at 37° C.Composition obtainable according to this method was used for theprophylaxis and treatment of interdigital and/or digital dermatitisand/or interdigital phlegmon in cattle as described below.

EXAMPLE 12

The species Candida albicans DSM-9456 is cultivated on agar Sabouraud,for example in 7 Roux flasks. Culture is cultivated for 7 days at 37° C.The blastospores are washed off with sterill water. The concentration ofblastospores in suspension is adjusted to 40 million per ml. Thehomogenate is inactivated by adding formaldehyde to reach 0.5% (v/v) incell suspension. The mixture is incubated for 7 days at 37° C.Composition obtainable according to this method was used for theprophylaxis and treatment of interdigital and/or digital dermatitisand/or interdigital phlegmon in cattle as described below.

EXAMPLE 13

A solution of chitosan modified by valeric acid chloride was added tothe vaccine Polivac-T against dermatophytosis of cattle (manufacturer:“Vetbiochim” LLC, Moscow; Distributor: “Prostore” LLC, Moscow) to reacha final concentration of 0.1% (w/v). The concentration of microconidiawas 40 million per ml. Composition obtainable according to this methodwas used for the prophylaxis and treatment of interdigital and/ordigital dermatitis and/or interdigital phlegmon in cattle as describedbelow.

EXAMPLE 14

Dermatophyte culture of the species Trichophyton verrucosum DSM-28406 iscultivated on agar/wort, for example in 7 Roux flasks. The culture iscultivated for 27 days at 26° C.

The fungal masses of the dermatophyte is lifted off and homogenised in500 ml an aqueous solution of 0.2% of chitosan modified byparaaminobenzoic acid. The concentration of microconidia is adjusted to50 million per ml for each homogenate. The homogenate is inactivated byadding formaldehyde to reach an end concentration of 0.5% (v/v) directlyto the cell suspension. The mixture is incubated for 7 days at 37° C.The composition obtainable according to this method was used for theprophylaxis and treatment of interdigital and/or digital dermatitisand/or interdigital phlegmon and/or dermatophytosis in cattle asdescribed below.

EXAMPLE 15

The species Candida albicans DSM-9456 is cultivated on malt extract-agarfor 6 Roux flasks. Culture is cultivated for 7 days at 35° C. Theblastospores are washed off with a physiological solution of sodiumchloride.

The fungal masses is lifted off and homogenised in an 500 ml aqueoussolution of 0.1% solution of chitosan modified by valeric acid chloride.The concentration of microconidia is adjusted to 80 million per ml inhomogenate. The homogenate is inactivated by adding formaldehyde toreach 0.2% (v/v) in end directly to the cell suspension. The mixture isincubated for 7 days at 37° C. Composition obtainable according to thismethod was used for the prophylaxis and treatment of interdigital and/ordigital dermatitis and/or interdigital phlegmon in cattle as describedbelow.

EXAMPLE 16

The fraction obtainable according to this process consists of antigenicnonsoluble material comprising polysaccharide and/or glycopeptides(ANMD) as disclosed in WO 97/07232. Dermatophyte culture of the speciesTrichophyton verrucosum DSM-28406 is cultivated on agar/wort, forexample in 10 Roux flasks. The culture is cultivated for 28 days at 28°C.

The resulting fungal biomass is lifted off and treated with an aqueoussolution of alkali. Preferred aqueous alkaline solutions are NaOH atconcentration of 3% (w/v). Alkaline treatment is preferably performed at80° C. for up to 6 h. Following the processing under aqueous alkalineconditions, the solid and liquid phases of the preparation are separatedby centrifugation at forces of about 3500 g. After alkaline treatment,the solid phase is treated with 0.2 M acetic acid are added to the solidphase for 1 hour at temperatures of 70° C. After acidic treatment thesolid phase is washed with distilled water. The washing is repeatedthree times. Finally, the solid phase is lifted off and homogenised in500 ml water for injection. The concentration of the particles isadjusted to 60 million per ml end product. Composition obtainableaccording to this method was used for the prophylaxis and treatment ofinterdigital and/or digital dermatitis and/or interdigital phlegmon incattle as described below.

EXAMPLE 17

The fraction obtainable according to this process consists of antigenicnonsoluble material comprising polysaccharide and/or glycopeptides(ANMP) as disclosed in WO 97/07232. Dermatophyte culture of the speciesTrichophyton verrucosum DSM-28406 is cultivated on agar/wort in 9 Rouxflasks. The culture is cultivated for 25 days at 27° C. The resultingfungal biomass is lifted off and treated with an aqueous solution ofalkali. Preferred aqueous alkaline solutions are KOH at preferredconcentrations of 2% (w/v). Alkaline treatment is preferably at 60° C.for up to 10 h. Following the processing under aqueous alkalineconditions, the solid and liquid phases of the preparation are separatedby centrifugation at forces of about 3500 g. The treatment under aqueousalkaline conditions was repeated two times, as well as the separationstep by centrifugation at forces of about 3500 g. After alkalinetreatment, the solid phase is treated with 0.5M HCl is added to thesolid phase for 1.5 hours at temperatures of 80° C. After acidictreatment the solid phase is washed with distilled water two times.Finally, the solid phase is lifted off and homogenised in 500 ml anaqueous solution comprising 0.3% chitosan modified by valeric acidchloride. The concentration of particles is adjusted to 60 million perml for each homogenate. The composition obtainable according to thismethod was used for the prophylaxis and treatment of interdigital and/ordigital dermatitis and/or interdigital phlegmon in cattle as describedbelow.

EXAMPLE 18

The product is prepared from Chitosan. The product is prepared in twostages. In the first stage a concentrated modified chitosan gel isobtained, in the second stage the final product, which can be used foradministration.

The First Step.

Chitosan with deacetylation of 80%, a viscosity of 2751-3250 mPas isused as raw material. 40 grams of the polysaccharide is sterilized byautoclaving and 3.5 liter of water for injection is added understirring. In the obtained suspension 40 ml of acetic acid are added. Thefinal volume is adjusted with water for injection to 4 liter. Suspendedpolysaccharide is stirred in a sterile container for 24 hours until agel suspension is obtained. Unsolved particles are removed by filtrationthrough a metal grid with a pore size of 200 μm-300 μm. 4 N sodiumhydroxide (NaOH) is added dropwise to the obtained suspension until thesuspension has a pH of 8.0. Upon that white flakes precipitatecomprising the chitosan. The suspension is stirred for 30 minutes. 8 mLlactic acid is added dropwise to the suspension under constant stirring.The obtained suspended material is stirred for another hour. Flakes andunsolved particles are separated from the suspension and resuspended in4 liter water for injection. 4N hydrochloric acid is added understirring until a pH of 5.6 is reached. The resultant suspension isdialysed in a closed system to remove free ions of salts and lowmolecular weight compounds. The resultant suspension is a concentratedmodified chitosan gel. The modified chitosan concentration is about 0.8%to about 1%. After the dialysis, the polysaccharide is used to preparethe final product.

The Second Step.

For obtaining the final product comprising the modified chitosanobtained in the first step the resultant suspension of the first step isadjusted to a volume of 25 liter by adding sterile water for injectionunder stirring. Then, 500 ml of chlorocresol solution containing 30grams chlorocresol are added to the mixture as preservative. Theresultant suspension is adjusted to a volume of 30 liter. The resultantsterile product is dispensed into vials under aseptic conditions.Composition obtainable according to this method was used for theprophylaxis and treatment of interdigital and/or digital dermatitisand/or interdigital phlegmon in cattle as described below.

EXAMPLE 19

Dermatophyte culture of the species Trichophyton verrucosum DSM-28406 iscultivated on agar/wort, for example in 8 Roux flasks. The culture iscultivated for 30 days at 28° C.

The fungal masses of the dermatophyte is lifted off and homogenised in500 ml an aqueous solution of 0.1% chitosan modified by valeric acidchloride. The concentration of microconidia is adjusted to 400 thousandper ml for each homogenate. The homogenates are inactivated by addingformaldehyde to reach 0.4% (v/v) in end directly to the cell suspension.The mixture is incubated for 7 days at 37° C. Composition obtainableaccording to this method was used for the prophylaxis and treatment ofinterdigital and/or digital dermatitis and/or interdigital phlegmonand/or dermatophytosis in cattle as described below.

EXAMPLE 20

Dermatophyte culture of the species Trichophyton mentagrophytes DSM-7279is cultivated on agar/wort in 6 Roux flasks. The culture is cultivatedfor 20 days at 26° C.

The fungal masses of the dermatophyte is lifted off and homogenised in400 ml an aqueous solution of 0.2% chitosan modified by paraaminobenzoicacid. The concentration of microconidia is adjusted to 250 thousand perml for each homogenate. The homogenates are inactivated by addingformaldehyde to reach 0.3% (v/v) in end directly to the cell suspension.The mixture is incubated for 6 days at 37° C. Composition obtainableaccording to this method was used for the prophylaxis and treatment ofinterdigital and/or digital dermatitis and/or interdigital phlegmonand/or dermatophytosis in cattle as described below.

EXAMPLE 21

Cows with clinical evidence of lameness and lesions of the interdigitalspace, which are typical for DD, ID and IP, were treated with variouscompositions. The composition was administered by intramuscularinjection two times with an interval of 10 days. The dose forcomposition was 5 ml for each application.

The results are shown in Table 2.

Amount of healthy animals Composition Frequency of In 30-35 days In53-55 days No of as prepared in drug after the first after the firstgroup example No of animals administration application application 1 110 2 4 5 2 2 10 2 4 5 3 3 10 2 5 5 4 4 10 2 4 5 5 5 15 2 7 7 6 6 13 2 66 7 7 10 2 4 5 8 8 11 2 5 6 9 9 12 2 5 6 10 10 13 2 6 6 11 11 14 2 7 712 12 13 2 6 7 13 13 10 2 5 7 14 14 12 2 5 7 15 15 14 2 6 8 16 16 10 2 55 17 17 12 2 6 6 18 18 14 2 7 9

No common or local reactions after application of the compositions wereobserved. Therapeutic efficacies of treatment with differentcompositions produced as described in Examples 1 to 18 were 40% to 64%.

EXAMPLE 22

Cows with clinical evidence of lameness and lesions of the interdigitalspace, which are typical for DD, ID and IP, were treated with variouscompositions. The composition was administered by intracutaneousinjection two times with an interval of 7-10 days. The composition dosewas 0.4 ml in total which was injected into two sites of the animal.

The results are shown in Table 3.

Amount of healthy animals Composition as Frequency of In 30-35 days In53-55 days No of prepared in drug after the first after the first groupexample No of animals administration application application 1 19 100 260 85 2 20 100 2 58 87

No common or local reactions after application of the compositions wereobserved. Therapeutic efficacy of treatment with different compositionsproduced according to examples 19 and 20 were about 85% to 87%.

EXAMPLE 23

Cows with clinical evidence of lameness and lesions of the interdigitalspace, which are typical for DD, ID and IP, were treated with variousdrugs. The composition prepared in accordance with Example 15 wasadministered as follows: 2 times intramuscular in a dose of 5 ml at onesite with an interval of 10 days.

Clinical Manifestation of Disease:

+ Recovering, or gray, no pain++ in healing, <2 cm, yellow, light pain+++ Recovering, or >2 cm, yellow, moderate pain++++ acute disease>2 cm, red, significant pain

The results are shown in Table 4.

Before treatment 30 days after treatment 58 days after treatment 12animals ++ 4 animals-healthy 11 animals-healthy 4 animals + 3 animals +6 animals ++ 4 animals +++ 2 animals +++ 3 animals ++ 4 animals ++++ 2animals ++++ in total 2 animals were in total 3 animals were culledculled for slaughter for slaughter

No common or local reactions after application were observed. Efficacyof treatment was about 70%.

EXAMPLE 24: DOSE TITRATION STUDY

Cows with clinical evidence of lameness, lesions of the interdigitalspace, which are typical for DD, ID and IP, were treated with variousdrugs. Therapeutic application of the composition prepared in accordancewith Example 14: 3 times intramuscular at one site with an interval of 7days.

Clinical Manifestation of Disease:

+ Recovering, or gray, no pain++ In healing, <2 cm, yellow, light pain+++ Recovering, or >2 cm, yellow, moderate pain++++ acute disease>2 cm, red, significant pain

The results are shown in Table 5.

51 days Before after treatment Before 51 days after treatment treatmentDose - 5 ml treatment Dose - 3 ml 19 5 animals-healthy 18 1animal-healthy animals +++ 7 animals + animals +++ 4 animals + 5 animals++ 6 animals ++ 2 animals +++ 2 animals were 5 animals were culledculled for slaughter for slaughter

No common or local reactions after application were observed. Efficacyof treatment with dose 5 ml was about 63% and 3 ml-28%.

EXAMPLE 25

Cows with clinical evidence of lameness and lesions of the interdigitalspace, which are typical for DD, ID and IP, were treated. Therapeuticapplication of the composition prepared in accordance with Example 14:intramuscular, 2 times at one site in dose of 5 ml with an interval of10 days.

Summary of Investigation:

Before treatment Amount of animals/amount of limbs with lameness 100/13852 to 54 days after last treatment Amount of animals/amount of limbswith lameness 29/29 Amount of healthy animals 71 Degrease of limbs withlameness 78.99%

EXAMPLE 26—DOSE TITRATION STUDY

Treatment of cows against DD, ID and IP was done. Prophylacticapplication of a composition prepared in accordance with Example 14:intramuscular, 2 times with an interval of 10 days.

Clinical Manifestation of Disease was Investigated:

+ Recovering, or gray, no pain++ in healing, <2 cm, yellow, light pain+++ Recovering, or >2 cm, yellow, moderate pain++++ acute disease>2 cm, red, significant pain

The results are shown in Table 6.

73 days after application Dose 1 ml Dose 2.5 ml Control 100 animals 100animals 215 animals 7 animals + 7 animals + 21 animals + 18 animals ++ 6animals +++

No common and local reactions after application of the composition wereobserved. Efficacy of treatment with doses of 1 ml and 2.5 ml was about93%. 45 animals (about 21%) from control group were with clinicalsymptoms of DD, ID and IP.

The results are shown in Table 7.

107 days after application of composition Dose 1 ml Dose 2.5 ml Control100 animals 100 animals 215 animals 7 animals + 9 animals + 11 animals +12 animals ++ 4 animals-culling for 4 animals-culling for 9 animals +++slaughter slaughter 27 animals-culling for slaughter

Efficacy of treatment with doses of 1 ml and 2.5 ml was about 87%-89%.59 animals (about 27%) from control group were with clinical symptoms ofDD, ID and IP.

The results are shown in Table 8.

170 days after application of composition Dose 1 ml Dose 2.5 ml Control100 animals 100 animals 215 animals 25 animals + 41 animals + 112animals + 6 additional animals were 3 additional animals 42 additionalanimals culled for slaughter were culled for were culled for slaughterslaughter

The efficacy of treatment with doses of 1 ml was about 70% and withdoses of 2.5 ml was about 53%. 154 animals (about 72%) from the controlgroup were with clinical symptoms of DD, ID and IP. This investigationdemonstrates prophylactic treatment of animals with a dose of 1.0 ml.Duration of immunity was about 5.5 month.

EXAMPLE 27

Treatment of cows against DD, ID and IP was done. Prophylacticapplication of a composition prepared in accordance with Example 14:intramuscular, 2 times in a dose of 5 ml with an interval of 10 days.

Summary of Investigation:

Animals in-group 1 were treated Observation before the treatment Amountof animals/clinical manifestations of DD, ID and IP 100/100 160 to 175days after last treatment Amount of animals/amount of limbs withlameness 100/10 Amount of healthy animals 90 Efficacy of treatment 90%Animals in group 2 were treated with placebo (control) Observationbefore treatment Amount of animals/clinical manifestations of DD, ID andIP 100/100 160 to 175 days after last application of placebo Amount ofanimals/amount of animals with manifestations of 100/48  Clinicalsymptoms of DD, ID and IP Amount of healthy animals 52 Amount of illanimals during of observation time 48%

All animals of the control group (placebo) with clinical symptom ofdiseases were treated with local application of aseptic medicine orantibiotics. In case of IP the intramuscular injection of antibioticswas used.

EXAMPLE 28

Cows with clinical evidence of lameness and lesions of the interdigitalspace, which are typical for DD, ID and IP, were treated with variouscompositions. The composition was administered by intramuscularinjection two times at one site with an interval of 10 days. The dose ofthe composition was 5 ml for each application.

The results are shown in Table 9.

Amount of healthy animals Frequency of In 30-35 days In 53-55 days No ofComposition as No of drug after the first after the first group preparedin example animals administration application application 1 14 10 2 1010 2 15 10 2 9 9 3 18 10 2 10 10 4 Control - treated 10 — 3 5 by commonmethods

EXAMPLE 29. HYDRO-COLLOIDS

Chemical nomenclature: Chitosan-Valeric acid-Hydro-Colloid

Subtitle: Polyaminosugar-Valeric acid-Hydrocomplex

Structural formula:

(C₆H₁₁NO₄)_(x)(C₈H₁₃NO₅)_(y)(C₅H₁₀O₂)_(z)(HCl)_(z)(H₂O)_(m)  Chemicalformula:

General Properties

Molecular weight: x*(161)+y*(203)+z*(102)+z*(36.5)+m*(18)

Appearance: natural white to yellowish viscous liquid with typical odor

Solubility: soluble in: Water

Odor: typical, similar to Valeric acid

Density: 1.002

pH-value: 5.5

Storage: Keep protected from light; store in a container protected fromair in a refrigerator at 4°-8° C.

Stability: 36 months under conditions described above

Chemical nomenclature: Chitosan-4-Aminobenzoic acid-Hydro-Colloid

Subtitle: Polyaminosugar-p-Aminobenzoic acid-Hydrocomplex

Structural formula:

(C₆H₁₁NO₄)_(x)(C₈H₁₃NO₅)_(y)(C₇H₇NO₂)_(z)(H₂O)_(m)  Chemical formula:

General Properties

Molecular weight: x*(161)+y*(203)+z*(137.14)+m*(18)

Appearance: Yellowish to yellow viscous liquid

Chemical nomenclature: Chitosan-Glucuronic acid-Hydro-Colloid

Subtitle: Polyaminosugar-Glucuronic acid-Hydrocomplex

Structural formula:

(C₆H₁₁NO₄)_(x)(C₈H₁₃NO₅)_(y)(C₆H₁₀O₇)_(z)(H₂O)_(m)  Chemical formula:

General Properties

Molecular weight: x*(161)+y*(203)+z*(194.14)+m*(18)

Appearance: Yellowish to yellow viscous liquid

EXAMPLE 30. MANUFACTURING OF CHITOSAN-VALERIC ACID-HYDRO-COLLOID

Purification of Chitosan 80/100 and 80/200, AS-No.: 9012-76-4,

Amino-N-acetyl-D-glucosamine is sterilized in a separate vessel and iscarried out to obtain Chitosan in pharmaceutical quality.

Reagent Solution

Sterile Amino-N-acetyl-D-glucosamine is resuspended under stirring for15 minutes in this sterile water. 400 ml of Acetic acid is added tosuspension under stirring (24 h) until a clear solution is obtained.

Purification Step

To this solution the 4 N Sodium hydroxide solution is added drop by drop(carefully) to obtain a pH 8.0 to 8.5. The resulting solutionprecipitates to a white mass. The obtained suspension is stirred notless than 30 minutes. The residue is separated from the liquid phase byfiltration.

Resuspension

The precipitate is resuspended in an equal amount of purified (sterile)water (water for injection (Pharm. Eur.)) (401, initial amount). 80 mlof Pentanoyl chloride is measured. Under stirring conditions thePentanoyl chloride is added drop by drop to the suspension. The obtainedsuspension is stirred until the solution is clear. 1.6 g Thiomersal isadded (40 m/mL). The clear solution is the active ingredient(Hydro-Colloid). The obtained polysaccharide colloid (CVHC) is storedunder 4° C. to 8° C. For an end product a aqueous solution is done withdefined biological activity.

Overview of the Reaction Steps of Manufacturing 1.Chitosan+water→suspension

suspension+HAc (24 h)→Chitosan-HAc-solution2. Purification step

2.1. Chitosan-HAc-solution+4N NaOH→(pH 8-8.5) Chitosan+NaAc+H₂O

2.2. Chitosan+NaAc+H₂O→H₂O+NaAc

-   -   →Chitosan (solid, purified)

3. Production

Chitosan (solid)+H₂O+Pentanoylchlorid→Chitosan+Valeric acid+H₂O+HCl→CVHC (Chitosan-Valeric acid-Hydro-Colloid)

The production is a combination of a purification step of the basicmaterial Chitosan and in process reaction with the second reagentPentanoyl chloride.

This first critical step is the precipitation of Chitosan to obtain thetotal amount of the purified chitosan in pharmaceutical quality.

In process control: The reaction time and the pH-value are monitored toget a quantitative precipitation.

Test for the pharmaceutical quality of chitosan:

Test for the quality of the intermediate (Chitosan pharm quality)

Solubility in water: A sample of about 250 mg of the precipitate ofChitosan is resuspended in 1 ml of purified water Target: No solubilitycan be obtained

Quality: is fulfilled if no reduction of the amount of the solidmaterial can be detected.

Solubility in stronger acids: In parallel same amount of precipitate issuspended in 1 ml HCl (3N)

Target: Total solution

Quality: is fulfilled if a solution of the total amount of the solidmaterial can be detected.

The second critical step is the dissolution process to the activeingredient. The control is done visually: The total amount of theprecipitate should be solubilized.

EXAMPLE 31. EXAMINATION ON IDENTITY BY USING UV/VIS-SPECTROSCOPY

Test method according to EUROPEAN PHARMACOPOEIA 2.2.25 was used.

Apparatus: Spectrophotometer Jasco 7800

Conditions of measurement: Bandwidth 2 nm

-   -   Range 200-600 nm    -   Blank correction with solvent    -   Temperature: 25° C.    -   UV-Cell: 12.5×45 mm semi-micro, 10 mm path length UV-grade        silica

Solvent: H₂O

Test solution: An adequate sample of Chitosan HCl, Chitosan-HAc,Chitosan, Chitosan-Valeric acid-Hydro Colloid and valeric acid,respectively was dissolved in the solvent above. This mixture wasshacked and afterwards sonified in an ultrasonic bath for 5 min.

The absorption maxima according to the general fundamentals ofspectroscopy and the chemical structure with specific chromophore groupsand substituents can be expected at: 200 nm for Chitosan HCl,Chitosan-HAc, Chitosan-Valeric acid-Hydro Colloid and valeric acid,respectively

UV- Chitosan Chitosan Maximum HCI HAc Chitosan CVHC Valeric acid nm 200200 — 200 211

The absorption maxima of Chitosan could not be analysed since Chitosanis a water insoluble solid, which can also not be solubilized in typicalorganic solvents.

The comparison of all spectra show no significance or structuralmodification like aromatic bonds etc. Based on the measured spectra andliterature data of the raw materials the measured spectrum correspondsto prospected spectra. Thus, the measured data above confirm theidentity of the prospected structure.

EXAMPLE 32: IR-ABSORPTION SPECTROPHOTOMETRY

Test method according to EUROPEAN PHARMACOPOEIA 2.2.24 was used.

For identification of the active principle Chitosan-Valericacid-Hydro-Colloid a series of IR-spectra of differentChitosan-Derivates are compared with the spectrum of the product and ofValeric acid.

1. Method and Parameters

Apparatus Infrared-Spectrometer FT/IR 410 Jasco

Range: 4000 cm⁻¹ to 600 cm⁻¹

Test sample: A mixture of 4.8 mg of Chitosan, or a mixture of 4 mg ofChitosan-HCl or a mixture of 3.8 mg of Chitosan Acetate and 100 mg KBris carefully grinded and pressed to a suitable potassium bromide disk,or a film of Chitosan-Valeric acid-Hydro Colloid or NaCl plate forvaleric acid

Conditions of Measurement:

Background correction: actual

Temperature 20° C.

The measured spectrum corresponds directly to the literature spectrafrom database.

Result: The measured data above confirms the identity of the testedsubstances.

2. Data of the Different IR-Spectra

Dried Chitosan- Chitosan- Chitosan- valeric acid Base HCl Chitosan-HAcColloid Valeric acid 3398 3365 3424 3426 2919/2875 2887 2926/ 2960-28722960-2875 2673 2018 2092 2130 1708 1717 1665 1596 1606 1562 1561 15691509 1467/1456 1421 1410 1408 1424 1413 1377 1380 1381 1320 1320 13361315 1279 1256 1246 1254 1236 1215 1154 1155 1155 1154 1079/1032 10841089 1076-1013 1109  897 896  890  926  940

The IR signals of Valerie acid in the active principle are very small tonot visible.

Comparison to literature data: Based on the measured spectra andliterature data of the raw materials, the measured spectrum of CVHCcorresponds to prospected spectrum.

Result: The measured data above confirm the identity of the proposedstructure.

EXAMPLE 33. ¹³C-NMR-SPECTROSCOPY ANALYSIS

Test method according to EUROPEAN PHARMACOPOEIA 2.2.33 was used.

a) 13C-NMR-Spectrum of Chitosan 1. Method and Parameters

Apparatus Bruker AMX 500 AVANCE

Conditions of measurement

-   Scan frequency: 125 MHz for Chitosan, Chitosan HCl, Chitosan HAc,    Glucosamin HCl, N-Acetylglucosamin, Chitosan-Valeric    acid-Hydro-Colloid, Valerie acid-   Temperature: 300 K for Chitosan, Chitosan HCl, Chitosan HAc,    Chitosan-Valeric acid-Hydro-Colloid, Valerie acid;    -   301 K for Glucosamin HCl and N-Acetylglucosamin-   Solvent: D₂O for Chitosan, Chitosan HCl, Chitosan HAc, Glucosamin    HCl, N-Acetylglucosamin;    -   DMSO-D6 for Chitosan-Valeric acid-Hydro-Colloid    -   CDCl₃ for Valerie acid-   Concentration: —for Chitosan, Chitosan HCl, Chitosan HAc,    Chitosan-Valeric acid-Hydro-Colloid;    -   approx. 15 mg/0.5 ml for Glucosamin HCl, N-Acetylglucosamin and        Valerie acid-   Calibration: —for Chitosan, Chitosan HCl, Chitosan HAc, Glucosamin    HCl, N-Acetylglucosamin    -   DMSO-D6 for Chitosan-Valeric acid-Hydro-Colloid    -   CDCl₃ for Valerie acid

2. Results

a)¹³C-NMR-Spectroscopy Analysis of Chitosan

Measurement in solution: According to the missing solubility in neutralsolvents a measurement in solution is not possible.

Measurement in solid state: A measurement in solid state was notpossible. Also after long measurement conditions (time) no acceptablesignals appeared.

Result: NMR-Identification of Chitosan is not possible.

b)¹³C-NMR-Spectroscopy Analysis of Chitosan HCl

Classification Results [d] (Carbon number) 97.67 C1 76.41/74.80 C5 70.28C3 64.41 C4 60.11 C6 56.06 C2 [ppm] Target The following characteristicchemical shifts 100 according to the general fundamentals of 70spectroscopy and the chemical skeleton with 56 substituents can beexpected at: General Literature: Hesse, Meier, Zeeh Spektr. MethodenThieme Verlag 5. Auflage

Result: The measured data above confirms the identity of the testedsubstance.

c)¹³C-NMR-Spectroscopy Analysis of Chitosan HAc

Classification Results [d] (Carbon number) Glucosamine 98.39 C1 skeleton74.79 C5 — C3 — C4 — C6 — C2 Acetic Acid 23.82 CH₃ 180.31 >C═O [ppm]Target The following characteristic chemical shifts 98.39 according tothe general fundamentals of spe spectroscopy and the chemical skeletonwith 23.82 substituents can be expected at: 180.31 General Literature:Hesse, Meier, Zeeh Spektr. Methoden Thieme Verlag 5. Auflage

Result: The measured data above confirms the identity of the testedsubstance.

d)¹³C-NMR-Spectroscopy Analysis of Glucosamin HCl

Classification Results [d] (Carbon number) 92.94/89.34 C1 76.25 C572.28/71.69 C3 69.85/69.77 C4 60.66/60.51 C6 54.62/57.08 C2 [ppm] TargetThe following characteristic chemical shifts 92.94/89.34 according tothe general fundamentals of 60.66/60.51 spectroscopy and the chemicalskeleton with 54.62/57.08 substituents can be expected at: GeneralLiterature: Hesse, Meier, Zeeh Spektr. Methoden Thieme Verlag 5. Auflage

Comparison to literature data: The measured spectrum correspondsdirectly to the literature spectra from database.

Result: The measured data above confirms the identity of the testedsubstance.

e)¹³C-NMR-Spectroscopy Analysis of N-Acetylglucosamin

Classification Results [d] (Carbon number) 95.06/90.95 C1 76.01/74.08 C571.64/70.86 C3 70.22/69.99 C4 60.89/60.74 C6 56.90/54.26 C2 22.29/22.03CH₃ 174.85/174.59 C═O Target The following characteristic chemicalshifts 95.06/90.95 according to the general fundamentals of 22.29/22.03spectroscopy and the chemical skeleton with 174.85/174.59 substituentscan be expected at: General Literature: Hesse, Meier, Zeeh Spektr.Methoden Thieme Verlag 5. Auflage

Result: The measured data above confirms the identity of the testedsubstance.

f)¹³C-NMR-Spectroscopy Analysis of Chitosan-Valeric Acid-Hydro-Colloid

Classification Results [d] (Carbon number) Glucosamine 100.95 C1skeleton 78.57 C5 76.10 C3 73.10 C4 61.40 C6 57.57 C2 Valeric acid180.66  C5{acute over ( )} 29.23  C4{acute over ( )} 24.85  C3{acuteover ( )} 23.37  C2{acute over ( )} 14.87  C1{acute over ( )} [ppm]Target The following characteristic chemical shifts 100.95 according tothe general fundamentals of 61.40 spectroscopy and the chemical skeletonwith 57.57 substituents can be expected at: 180.66 14.87 GeneralLiterature: Hesse, Meier, Zeeh Spektr. Methoden Thieme Verlag 5. Auflage

Result: The measured data above confirms the identity of the proposedstructure.

g)¹³C-NMR-Spectroscopy Analysis of Valeric Acid

Classification Results [d] (Carbon number) 180.5 C5 33.8 C4 26.7 C3 22.2C2 10.6 C1 [ppm] Target The following characteristic chemical shifts 180according to the general fundamentals of 10.6 spectroscopy and thechemical skeleton with substituents can be expected at: GeneralLiterature: Hesse, Meier, Zeeh Spektr. Methoden Thieme Verlag 5. Auflage

Comparison to literature data: The measured spectrum correspondsdirectly to the literature spectra from database.

Result: The measured data above confirms the identity of the testedsubstance.

3. Comparison of the NMR Spectra

Gluco- N- Chitosan- Classification Chitosan Chitosan samin Acetyl-Valeric acid- Valeric (Carbon HCl HAc HCl glucosamin Hydro- acid number)97.7 98.4 92.9/89.3 95.0/91.0 101.0 C 56.1 54.6/57.1 54.3/56.9 57.6 C70.3 71.7/72.3 71.6/70.9 76.1 C 64.4 69.9/69.8 70.2/70.0 73.1 C76.4/74.8 74.8 7 76.0/74.1 78.6 C 60.1 60.5/60.7 60.9/60.7 61.4 C 22.723.8 180.3 174.9/174.6 14.9 13.0 23.4 22.0 24.9 26.0 29.2 33. 108.7 180.Literature — — X — — X

Comparison to literature data: Not available or Based on the measuredspectra and literature data of the raw materials, the measured spectrumcorresponds directly to prospected spectra. Result: The measured dataabove confirm the identity of the proposed structure.

EXAMPLE 34. TLC-METHOD FOR THE ANALYSIS OF CHITOSAN AND IMPURITIES INTHE NEW PRODUCT CHITOSAN-VALERIC ACID-HYDRO-COLLOID (CVHC)

Test method according to EUROPEAN PHARMACOPOEIA 2.2.27 was used.

This part presents the procedures and data of thin layer chromatographyfor the identification of CVHC along with the Rf values in the usedsolvent mixtures and spot colors when detected under UV-light (365 nmand 254 nm), visible light and with typical visualisation reagents.

The original based raw material for any kind of glucosamines is thenatural material Chitin from insects or crabs. The monomeric structureof these biopolymers is N-Acetyl-Glucosamine.

For pharmaceutical and other use in most cases deacetylated Chitin istypical. This resulting biopolymer is the so called Chitosan, which canbe modified into water soluble ionic compounds. The monomeric structureof this Chitosan should be theoretically Glucosamine. Because thedeacetylation step does not run totally, Chitosan has a mixed structureof N-Acetyglucosamine (acetylated) and Glucosamine (deacetylated) units.Chitosan-Valeric acid-Hydro-Colloid is a new Polyaminosugar-valeric acidhydro-complex. Therefore no positive analytical test results forN-Acetyl-Glucosamine and Glucosamine should be possible. If monomericfragments are embedded as residual impurities, it should be possible toidentify Chitosan in form of its water soluble ionic compounds ChitosanHCl and Chitosan HAc.

1. Method

Apparatus Camag Chromatographic Tank System TLC-plate Merck Si 60 F 254precoated plates Conditions Protected from sunlight and with chambersaturation Temperature 20-25° C. Development: Vertical development

Chromatographic Conditions

Sample-solution See the single analytes Application 30 μl Drying Min. 2minutes in an air-stream Motion range 80 mm

Mobile Phase

Solvents Acetone Water 25% aq. Ammonia — Mixture 20 10 5 —

2. Analysis and Results

a) Chitosan Sample Preparation

Sample: Chitosan suspended in water1) Apparatus: reflux condensorConditions: heating for about 30 minutes under reflux (145° C.)2) Apparatus: Ultra sonic bathConditions: Sonification for about 30 minutes at 45° C.3) Apparatus: reflux condensorConditions: heating for about 30 minutes under reflux (145° C.)4) Filtration: 0.45 μm filterThe clear filtrate was used for analysis.

Detection with UV-Fluorescence and VIS

Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No NoImpurities No No No

Detection with Visualisation Reagents

Group Group Anisaldehyde- specific specific Sulfuric acid Visible lightreagent 1 reagent 2 reagent Iodine Compound signal No No No NoImpurities No No No No Group specific reagent 1:Naturstoff-Reagent/DT/366 nm Group specific reagent 2: 5%Ninhydrine/EtOH Rf-value No signal for chitosan can be identified — Nonspecified impurities: Not detected

Alternative: Solubilization in organic solvents show equal resultsbecause of the missing solubility of Chitosan.

Result: An acceptable solution of Chitosan in waterish or organicsolvents like Methanol etc. is not possible. A suitable solubilizationof Chitosan is only possible in stronger acids like HCl or HAc underproduction of Chitosan HCl or Chitosan HAc.

b) Chitosan HCl

5 mg Chitosan HCl/ml H₂O was used for analysis.

Detection with UV-Fluorescence and VIS

Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No NoImpurities No No No

Detection with Visualisation Reagents

Group specific Group Anisaldehyde- reagent 1 specific Sulfuric acidVisible light reagent 2 reagent Iodine Compound No No Grey spot Brownspot signal Impurities No No No No Group specific reagent 1:Naturstoff-Reagent/DT/366 nm Group specific reagent 2: 5%Ninhydrine/EtOH Rf-value Chitosan HCl 0.0 Non specified impurities: Notdetected Target Compound purity One main spot Anisaldehyd-Sulfuricacid-reagent respectively Iodine as non-selective reagents for detectionof non-specified impurities should show no greater impurities A relativeretardation factor (Rf) of this compound according to the chemicalskeleton under this described chromato-graphic conditions for such anpolymer can be expected at: 0.0 — —

Result: Compound purity; One main spot.

c) Chitosan HAc

5 mg Chitosan HAc/ml H₂O was used for analysis.

Detection with UV-Fluorescence and VIS

Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No NoImpurities No No No

Detection with visualisation reagents

Group Group Anisaldehyde- specific specific Sulfuric acid Visible lightreagent 1 reagent 2 reagent Iodine Compound signal No No Grey spot Brownspot Impurities No No No No Group specific reagent 1:Naturstoff-Reagent/DT/366 nm Group specific reagent 2: 5%Ninhydrine/EtOH Rf-value Chitosan HAc 0.0 Non specified impurities: Notdetected Literature Value Not available data from — Target Compoundpurity One main spot Anisaldehyd-Sulfuric acid-reagent respectivelyIodine as non- selective reagents for detection of non-specifiedimpurities should show no greater impurities A relative retardationfactor (Rf) of this compound according to the chemical skeleton underthis described chromatographic conditions for such an polymer can beexpected at: 0.0 — —

Result: Compound purity; One main spot.

d) Glucosamine HCl

5 mg Glucosamine HCl/ml H₂O was used for analysis.

Detection with UV-Fluorescence and VIS

Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No NoImpurities No No No

Detection with Visualisation Reagents

Group Anisaldehyde- Group specific specific Sulfuric acid Visible lightreagent 1 reagent 2 reagent Iodine Compound Blue spot Red spot Grey spotBrown spot signal Impurities No No No No Group specific reagent 1:Naturstoff-Reagent/DT/366 nm Group specific reagent 2: 5%Ninhydrine/EtOH Rf-value Glucosamine HCl 0.67 Non specified impurities:Not detected Literature Not data from — Value available Target CompoundOne main spot purity Anisaldehyd-Sulfuric acid-reagent respectivelyIodine as non-selective reagents for detection of non-specifiedimpuritiesshould show no greater impurities A relative retardationfactor (Rf) of this compound according to the chemical skeleton underthis described chromato-graphic conditions can be expected between 0.6and 0.8

Result: Compound purity; One main spot.

e) N-Acetylglucosamine

5 mg N-Acetylglucosamine/ml H₂O was used for analysis

Detection with UV-Fluorescence and VIS

Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No NoImpurities No No No

Detection with Visualisation Reagents

Group Anisaldehyde- Group specific specific Sulfuric acid Visible lightreagent 1 reagent 2 reagent Iodine Compound Blue spot No Grey spot Brownspot signal Impurities No No No No Group specific reagent 1:Naturstoff-Reagent/DT/366 nm Group specific reagent 2: 5%Ninhydrine/EtOH Rf-value N-Acetylglucosamine 0.72 Non specifiedimpurities: Not detected Target Compound One main spot purityAnisaldehyd-Sulfuric acid-reagent respectively Iodine as non-selectivereagents for detection of non-specified impurities should show nogreater impurities A relative retardation factor (Rf) of this compoundaccording to the chemical skeleton under this described chromato-graphicconditions can be expected between 0.6 and 0.8

Result: Compound purity; One main spot.

f) Chitosan-Valeric acid-Hydro-Colloid (CVHC)

CVHC is a high viscous waterish gel. Two drops of CVHC was used foranalysis.

Detection with UV-Fluorescence and VIS

Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No NoImpurities No No No

Detection with Visualisation Reagents

Group Anisaldehyde- Group specific specific Sulfuric acid Visible lightreagent 1 reagent 2 reagent Iodine Compound No No Grey spot Brown spotsignal Impurities No No No No Group specific reagent 1:Naturstoff-Reagent/DT/366 nm Group specific reagent 2: 5%Ninhydrine/EtOH Rf-value Chitosan-Valeric acid-Hydro-Colloid 0.0 Nonspecified impurities: Not detected Literature Not available data from —Value Target Compound One main spot purity Anisaldehyd-Sulfuricacid-reagent respectively Iodine as non-selective reagents for detectionof non-specified impurities should show no greater impurities A relativeretardation factor (Rf) of this compound according to the chemicalskeleton under this described chromato-graphic conditions for such anpolymer can be expected at: 0.0 — —

Result: Compound purity; One main spot.

g) Valeric Acid

1 μl Valeric acid (pure) was used for analysis.

Detection with UV-Fluorescence and VIS

Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No NoImpurities No No No

Detection with Visualisation Reagents

Group Anisaldehyde- Group specific specific Sulfuric acid Visible lightreagent 1 reagent 2 reagent Iodine Compound No No No Yellow spot signalImpurities No No No No Group specific reagent 1:Naturstoff-Reagent/DT/366 nm Group specific reagent 2: 5%Ninhydrine/EtOH Rf-value Valeric acid 0.0 Non specified impurities: Notdetected Literature Not available data from — Value Target Compound Onemain spot purity Anisaldehyd-Sulfuric acid-reagent respectively Iodineas non-selective reagents for detection of non-specified impuritiesshould show no greater impurities A relative retardation factor (Rf) ofthis compound according to the chemical skeleton under this describedchromato-graphic conditions can be expected between 0.0 — —

Result: Compound purity; One main spot.

3. Comparison of the Results of the TLC Analysis

Gluco- N-Acetyl- Chitosan- Chitosan Chitosan samine glucoamine ValerieValerie Chitosan HCl HAc HCl HCl colloid acid Rf-value Not 0 0 0.67 0.720 0 possible Detection Compound signal UV 254 nm — — — — — — — UV 365 nm— — — — — — — Visible light — — — — — — — Naturstoff-Reagent — — — Bluespot Blue spot — — Ninhydrine — — — Red spot — — — Reagent Anisalde- —Grey Grey Grey Grey Grey — hyde-Sulfuric spot spot spot spot spot acidreagent Iodine — Brown Brown Brown Brown Brown Yellow Reagent spot spotspot spot spot Spot

The results above from TLC show that there is no evidence of monomericor dimeric structure which could be detected with the specificderivation reagents tested above. The detection and the Rf value of “0”show the similarity of Chitosan-Valeric acid-Hydro-Colloid to therelated compounds Chitosan HCl and Chitosan HAc. A specificidentification of Valeric acid with this TLC-System failed.Chitosan-Valeric acid-Hydro-Colloid can only be aPoly-Amino-sugar-colloid, but not a solution of Chitosan or a Chitosanderivate with Valeric acid in water.

EXAMPLE 35. TLC-METHOD FOR THE ANALYTICAL DETECTION OF VALERIC ACID INCHITOSAN-VALERIC ACID-HYDRO-COLLOID 1. Method and Parameters

A new TLC system was established for a identification and purity testingof the constituent Valeric acid.

Apparatus Camag Chromatographic Tank System TLC-plate Merck Si 60 F. 254precoated plates Conditions Protected from sunlight and with chambersaturation Temperature 20-25° C. Development: Vertical development

Chromatographic Conditions

Drying Min. 2 minutes in an air-stream Motion range 80 mm

Mobile Phase

Solvents Ethyl Acetate — — — Mixture 100 — — —

2. Results a) Valeric Acid (Pure)

2 μl of Valeric acid (pure) was used for analysis.

Detection with UV-Fluorescence and VIS

Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No NoImpurities No No No

Detection with Visualisation Reagents

Group specific Anisaldehyde-Sulfuric Visible light reagent acid reagentIodine Compound signal Yellow spot/blue Pink spot Yellowish backgroundspot Impurities No No No Group specific reagent: BromcresolGreen/Bromphenol Blue/Potassium Permanganate Reagent [Jork et al.]Rf-value Valeric acid 0.56 Non specified impurities: Not detectedLiterature Value Not available data from —

Detection limit: of valeric acid with this visualisation reagent afterTLC-chromatography: 0.03 μg

b) Chitosan-Valeric Acid-Hydro-Colloid (CVHV)

45 μl Chitosan-Valeric acid-Hydro-Colloid, pure (this is an about 850times higher amount of valeric acid, compared with the tests before) wasused for analysis.

Detection with UV-Fluorescence and VIS

Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No NoImpurities No No No

Detection with Visualisation Reagents

Group specific Anisaldehyde-Sulfuric Visible light reagent acid reagentIodine Compound signal Blue spot/blue Grey spot Brown spot backgroundImpurities No No No Group specific reagent: Bromcresol Green/BromphenolBlue/Potassium Permanganate Reagent [Pork et al.] Target Compound Onemain spot purity Anisaldehyd-Sulfuric acid-reagent respectively Iodineas non-selective reagents for detection of non-specified impuritiesshould show no greater impurities The Group specific reagent BromcresolGreen/ Bromphenol Blue/Potassium Permanganate Reagent should showtypical results for the compounds A relative retardation factor (Rf) ofthis compound according to the chemical skeleton under this describedchromatographic conditions can be expected at 0.0 forChitosan-derivatives appr. 0.6 for valeric acid if available Rf-valueChitosan-Valeric acid-Hydro-Colloid 0.0 Non specified impurities: Notdetected Rf-value Valeric acid Not detected Non specified impurities:Not detected Literature Value Not available data from —

Detection limit of valeric acid with this visualisation reagent afterTLC-chromatography: 0.03 μg

Pure Valeric acid can be identified with this TLC-System. Colloidalintegrated Valeric acid can not be detected in the pure compoundChitosan-Valeric acid-Hydro-Colloid. The detection and the Rf value of“0” show the similarity of Chitosan-Valeric acid-Hydro-Colloid to otherrelated Chitosan compounds. Chitosan-Valeric acid-Hydro-Colloid can onlybe a Poly-Amino-sugar colloid, but not a solution of Chitosan or aChitosan derivate with Valeric acid in water. The results above confirmthe identity of the proposed structure.

EXAMPLE 36. ELIMINATION OF VALERIC ACID FROM CHITOSAN-VALERICACID-HYDRO-COLLOID WITH HIGH VACUUM AND HIGHER TEMPERATURE

Method: Estimation of the loss on drying (special method)

Apparatus: Speed circulating vacuum concentrator

Conditions: 5 mbar

Temperature: 60° C.

Time: 1 week

End point: Constant mass

Appearance: Glassy mass

Result Odor: No typical odor from valeric acid

Sample Preparation

Redissolution partly with water

Appearance: High viscous gel

TLC-Analysis

Apparatus: Camag Chromatographic Tank System

TLC-plate: Merck Si 60 F 254 precoated plates

Conditions: Protected from sunlight and with chamber saturation

Temperature: 20-25° C.

Development: Vertical development

Chromatographic Conditions

Sample-solution See above

Application: 5 μl

Drying: Min. 2 minutes in an air-stream

Motion range: 80 mm

Mobile Phase

Solvents Ethyl Acetate — — — Mixture 100 — — —

Detection with UV-Fluorescence and VIS

Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No NoImpurities No No No

Detection with Visualisation Reagents

Group specific Anisaldehyde-Sulfuric Visible light reagent acid reagentIodine Compound signal Blue spot/blue grey spot brown spot backgroundImpurities No No No Group specific reagent: Bromcresol Green/BromphenolBlue/Potassium Permanganate Reagent [Jork et al.] Rf value spot 0

Detection limit: of Valeric acid with this visualization reagent afterTLC-chromatography: 0.03 μg

Result: With high vacuum and higher temperature a disproportion ofChitosan-Valeric acid-Hydro-Colloid takes place. The elimination ofValeric acid can be shown by absolutely no typical odor from Valericacid. The elimination of Valeric acid can be shown by TLC analysis: notypical spot of free valeric acid at Rf-value 0.56. Chitosan or Chitosancompounds can be identified at Rf-value 0. Chitosan-Valericacid-Hydro-Colloid can only be a Poly-Amino-sugar-colloid, but not asolution of Chitosan or a Chitosan derivate with valeric acid in water.

EXAMPLE 37. DISPROPORTION OF CHITOSAN-VALERIC ACID-HYDRO-COLLOID WITHSOLVENTS

The structure of Chitosan-Valeric acid-Hydro-Colloid is decomposed inEthyl acetate to Valeric acid and a Chitosan compound.

Sample Preparation

-   Apparatus: separating funnel, evaporator-   Liquid-liquid distribution: 20 ml Chitosan-Valeric    acid-Hydro-Colloid and 10 ml Ethyl acetate-   Conditions: Shaking for about 5 minutes and wait for phase    separation-   Separation of phases: The ethyl acetate phase was collected-   Concentration step: The about 10 ml were concentrated to liquid    residue (waterish) with an evaporator-   Resolubilization: in 1 ml Methanol-   Homogenization: Centrifugation step about 5 min 12.000 rpm-   Phase separation: Upper phase: clear methanolic solution    -   Lower phase: high viscous gel

TLC Analysis of Upper and Lower Phase (See Above)

a) Analysis of upper phase (clear methanolic solution)

TLC-Analysis

Apparatus: Camag Chromatographic Tank System

TLC-plate: Merck Si 60 F 254 precoated plates

Conditions: Protected from sunlight and with chamber saturation

Temperature: 20-25° C.

Development: Vertical development

Chromatographic Conditions

Sample-solution: clear methanolic solution

Application: 5 μl

Drying: Min. 2 minutes in an air-stream

Motion range: 80 mm

Mobile Phase

Solvents Ethyl Acetate — — — Mixture 100 — — —

Detection with UV-Fluorescence and VIS

Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No NoImpurities No No No

Detection with Visualisation Reagents

Group specific Anisaldehyde-Sulfuric Visible light reagent acid reagentIodine Compound signal Yellow spot/blue No Light background yellowishspot Impurities No No No Group specific reagent: BromcresolGreen/Bromphenol Blue/Potassium Permanganate Reagent [Jork et al.]Rf-value Valeric acid 0.57 Non specified impurities: —

Detection limit with visualisation reagent: 0.03 μg

Result: The upper phase is a clear methanolic solution. Valeric acid canbe identified after decomposition of the Hydro-Colloid in this solutionwith TLC. No Chitosan or Chitosan compound can be detected with TLC.

b) Analysis of Lower Phase (High Viscous Gel) TLC-Analysis

Apparatus: Camag Chromatographic Tank System

TLC-plate: Merck Si 60 F 254 precoated plates

Conditions: Protected from sunlight and with chamber

Temperature: 20-25° C.

Development: Vertical development

Chromatographic Conditions

Sample-solution: high viscous gel, totally redissolved in water

Application: 30 μl

Drying: Min. 2 minutes in an air-stream

Motion range: 80 mm

Mobile Phase

Solvents Ethyl Acetate — — — Mixture 100 — — —

Detection with UV-Fluorescence and VIS

Fluorescence wavelength 254 nm 365 nm VIS Compound signal No No NoImpurities No No No

Detection with Visualisation Reagents

Group specific Anisaldehyde-Sulfuric Visible light reagent acid reagentIodine Compound signal Blue spot/blue Grey spot Brown spot backgroundImpurities No No No Group specific reagent: Bromcresol Green/BromphenolBlue/Potassium Permanganate Reagent [Jork et al.] Rf-value Spot 0 Nonspecified impurities: —

Detection limit with visualisation reagent: 0.03 μg

Results: The lower phase is a high viscous gel, soluble in water. NoValeric acid can be detected in this phase by TLC. Chitosan or aChitosan compound can be identified in the lower phase (gel) by TLC.

Results A disproportion of Chitosan-Valeric acid-Hydro-Colloid is fromTLC possible with typical solvents like Ethyl acetate and analysisafterwards with Methanol A re-solubilization of from disproportionedChitosan-Valeric acid-Hydro- Colloid can be realized with Methanol Thedecomposition of Chitosan-Valeric acid-Hydro-Colloid in Ethyl acetateshows two phases Upper phase Lower phase Ethyl acetate phase AqueousColloid residue After concentration the Ethyl acetate phase wasredissolved in Methanol and results also two phases Upper phase Lowerphase clear methanolic solution high viscous gel This gel can bere-dissolved totally in water can be identified No can be identified NoChitosan or Chitosan Chitosan or a Chitosan compound can be detectedcompound can be detected

Summary of the Results

Chitosan-Valeric acid- Chitosan- Hydro-Colloid Valeric Elimination(decomposed with Ethyl Acid-Hydro- of from acetate) Colloid CVHC withUpper Lower Valeric acid (pure high phase phase pure CVHC) vacuum(Methanol) (Water) Rf-value 0.56 + − − + − Rf-value 0 − + + − +Detection Compound signal UV 254 nm — — — — — UV 365 nm — — — — —Visible light — — — — — Anisaldehyde-Sulfuric Pink spot Grey spot Greyspot — Grey spot acid reagent Iodine reagent Yellowish Brown Brown LightBrown spot spot spot yellowish spot spot Bromcresol Green Yellow spotBlue spot Blue spot Yellow spot Blue spot Bromphenol Blue reagent

Result: Chitosan-Valeric acid-Hydro-Colloid can only be aPoly-Amino-sugar-colloid, but not a solution of Chitosan or a Chitosanderivate with valeric acid in water. The results above confirm theidentity of the proposed structure.

EXAMPLE 38. ESTIMATION OF THE RELATIVE DENSITY

Because of the high viscosity of Chitosan-Valeric acid-Hydro-Colloid theestimation of the density is not possible with a densitybottle/pycnometer according to Test method according to EUROPEANPHARMACOPOEIA 2.2.5.

1. Test Method by Weighing

Apparatus: 250 ml volumetric flask

-   -   Balance: Sartorius MC 1 LC 2200S

Thermometer: Thermometer with graduation (min 0.5° C.) and a range notmore than 60° C.

Results 1.001 [d₂₀ ²⁰]

The active principle is a hydrogel, so the theoretical density should behigher than 1.0. The measured data confirms the identity of the proposedsubstance.

2. Test Method with Hydrometer

Test method according to EUROPEAN PHARMACOPOEIA 2.2.5 was used.

Apparatus: 250 ml volumetric flask

Hydrometer: Widder 1573°, 20° C.-M100-DIN 12791 Klasse H

Thermometer Thermometer with graduation (min 0.5° C.) and a range notmore than 60° C.

Conditions of measurement: Temperature 20+/−0.5° C. with electronicthermostate

Results 1.002 [d₂₀ ²⁰]

The active principle is a hydrogel, so the theoretical density should behigher than 1.0. The measured data confirms the identity of the proposedsubstance.

EXAMPLE 39. SULPHATED ASH

The Test method according to EUROPEAN PHARMACOPOEIA 2.4.14 was used.

Testing

Apparatus Suitable crucible (porcelain or platinum) were ignited at 600+/− 50° C. for 30 min in a “Muffel”-oven allow to cool in a desiccatorover silica gel or other suitable desiccant Estimation of crucibleweight Weight Weight of crucible 1: 52.0120 [g] Weight of crucible 2:57.6055 [g] Method 2 Additional for this Hydrogel a concentration stepto dryness (acid insoluble ash) was done by drying at 105° C. in annormal oven Sample: 25 ml_ of Hydrogel CVHC Usually 1-2 g Sample weight:usually 1-2 g or sufficient amount to obtain a residue of minimum 1 g.Moisten the sample with a small amount of sulfuric acid R [95-97% m/m](usuallyl ml_) and heat at as low temperature as practicable until theresidue is charred. After cooling, moisten the residue with a smallamount of sulfuric acid R [95-97% m/m](usuallyl ml_) Heat until whitefumes are no longer evolved Ignite at 600 +/− 50° C. for 30 min untilthe residue is completely incinerated. Flames are not allowed to beproduced at any time during the procedure allow to cool in a desiccatorover silica gel or other suitable desiccant Weigh and calculate thepercentage of residue Weighting of total Total Weight of crucible 1:52.0668 g weight Total Weight of crucible 2: 57.6612 g Sulphated ashValue 1: 0.0548 g content Value 2: 0.0557 g Average: 0.05525 g/25 ml_(—)Calculation of content of 0.05525 g/25.05 g = 0.0022055 g/g = sulphatedash 2.2055 mg/g 0.22%

EXAMPLE 40. LOSS ON DRYING

Based on this Phytochem® established appropriate methods for thedetermination of loss on drying.

1. Method and Parameter for Test of Chitosan HCl, Chitosan and ChitosanHAc Sample Preparation

Pretreatment of container: The substance is placed in a suitableweighing bottle, previously dried under the conditions used afterwords

Filling: the material is filled not higher than 5 millimeter

Transport: The weighing bottle is closed with a suitable cover

PC-method: A “under higher vaccum”

-   -   modified Pharmacopoeia-method 2.2.32 (EP) “in vacuum in a        desiccator”

Apparatus: desiccator

Drying time: to constant weight

Drying temperature: 25° C.±2° C.

Vacuum: permanent 4-8 mbar with specific pumps

Drying reagent: Diphosporuspentoxide (freshly)

2. Estimation of the Loss on Drying of Chitosan in Chitosan-ValericAcid-Hydro-Colloid (Special Method)

The content of Chitosan in Chitosan-Valeric acid-Hydro-Colloid isestimated with a gravimetric measurement.

Apparatus: Speed circulating vacuum concentrator

Method: Estimation of the loss on drying (special method)

Conditions of Measurement

Pressure: 5 mbar

Temperature: 60° C.

Time: 1 week

End point: Constant mass

Appearance: Glassy mass

Measurement: Test solution 4 ml Chitosan-Valeric acid-Hydro-Colloid

Repetition: 10 times

Result Odor: No typical odor from valeric acid

Weighing

1 40.20 mg 2 39.80 mg 3 40.20 mg 4 39.90 mg 5 40.10 mg 6 40.10 mg 739.90 mg 8 39.60 mg 9 40.20 mg 10 40.40 mg

Average 40.04 mg

Standard deviation 0.236643191

Relative standard deviation 0.591016961

Variance 0.056

Results: The weighing of the dried substance shows good similarity.Based on this measurements the content of Chitosan in Chitosan-Valericacid-Hydro-Colloid is 1%.

3. Comparison of the Results

Chitsoan Chitsoan Chitosan- Chitsoan HCI HAc Valeric acid- solid solidsolid Hydro-Colloid Loss on drying 7.2%  7.9% 20.3% . Residue from . . .1% drying Target EP . <10% . .

The active principle should be a Hydro Colloid gel. The measured dataconfirm the structure of compound.

EXAMPLE 41. ESTIMATION OF THE OSMOLARITY

The estimation of the Osmolarity can be done was an indirect measurementof the decrease of the melting point of a solution.

Apparatus: Halbmicro Osmometer Knauer

Conditions of measurement: External cooling system

Range: 0-1600 mOsmol

Method: Freezing

Test Procedure

Calibration with Standard solution 400 mOsmol/Kg: 12,687 g NaCl in 1 lWasser at 20° C.

Repetition: 2 times

Vessel: Specific glass vial

Sample: Chitosan-Valeric acid-Hydro-Colloid

Test solution: 1 without dilution

-   -   2 Dilution of 1:5

Quantity 150 μl each

Calibration

Sample Spezification Setpoint Measured value Calibration 1 Bidest. water 0 mOsmol  0 mOsmol Calibration 2 400 m Osmol/kg 400 mOsmol 400 mOsmol

Measurement

Number Sample Measured value 1a Chitosan-Valeric acid-Hydro-Colloid 100mOsmol 1b Chitosan-Valeric acid-Hydro-Colloid 110 mOsmol 2aChitosan-Valeric acid-Hydro-Colloid 1:5 20 mOsmol 2b Chitosan-Valericacid-Hydro-Colloid 1:5 20 mOsmol

Results: The measurement of the Osmorarity of Chitosan-Valericacid-Hydro-Colloid show a relatively low content. The measured contentof osmolar reacting components can only be so low, if there is nosolution or suspension of chitosans and valeric acid. The high viscousgelling compound can only be a Hydro-Colloid.

Result: The measured data above confirms the identity of the proposedsubstance.

1. A method comprising administering antigenic material ofkeratinophilic fungi and/or keratinophilic yeasts to treat or preventhoof- and claw diseases in animals, in particular in mammals, moreparticularly in bovidae and/or pigs, more particularly in cattle.
 2. Themethod of claim 1, wherein the antigenic material of keratinophilicfungi and/or yeasts comprise homogenised inactivated dermatophytemicroconidia and/or homogenised inactivated yeast blastospores and/orantigenic material of dermatophyte microconidia and/or antigenicmaterial of yeast blastospores.
 3. The method of claim 2, wherein theantigenic material of dermatophyte microconidia and/or antigenicmaterial of yeast blastospores comprise antigenic material, not solublein aqueous solutions, comprising polysaccharide and/or glycopeptide(ANMP), antigenic material, soluble in aqueous solutions, comprisingpolysaccharide and/or glycopeptide (ASMP) or antigenic exogenousmaterial comprising polysaccharide and/or glycopeptide (AEMP). 4-5.(canceled)
 6. The method of claim 1, wherein the antigenic material ofkeratinophilic fungi and/or keratinophilic yeasts are derived fromTrichophyton, in particular Trichophyton verrucosum, Trichophytonmentagrophytes, Trichophyton equinum, Trichophyton sarkisovii,Trichophyton rubrum or Trichophyton mentagrophytes, Microsporum, inparticular Microsporum gypseum, Microsporum canis, in particularMicrosporum canis var. obesum or Microsporum canis var. distortum and/orCandida, in particular Candida albicans.
 7. The of method of claim 1,wherein the antigenic material of keratinophilic fungi is derived fromthe strains Trichophyton mentagrophytes DSM-7279, Trichophytonverrucosum DSM-28406, Trichophyton rubrum DSM-9469, Trichophyton rubrumDSM-9470, Trichophyton rubrum DSM-9471, or Trichophyton rubrum DSM-9472and/or the antigenic material of keratinophilic yeasts is derived fromthe strains Candida albicans DSM-9456, Candida albicans DSM-9457,Candida albicans DSM-9458, or Candida albicans DSM-9459.
 8. The methodof claim 2, wherein the microconidia are in a swollen condition and/orhave germ tubes.
 9. The method of claim 2, wherein the blastosporesand/or microconidia have been inactivated with thiomersal, formaldehydeand/or 2-propiolactone.
 10. The method of claim 1, wherein thecomposition additionally comprises one or more immunomodulatorysubstance/s.
 11. The method of claim 1, wherein the compositioncomprises additionally chitosan modified by valeric acid, lactic acid,para-aminobenzoic acid, glucuronic acid or chloride of valeric acid or ahydro colloid comprising: (i) 0.1% to 5% (w/v) chitosan and 0.001 to 5%(w/v) valeric acid, or a salt thereof, preferably chloride of valericacid or (ii) 0.1% to 5% (w/w) chitosan and 0.001 to 5% glucuronic acidor (w/w) p-aminobenzoic acid or a salt thereof.
 12. The method of claim1, wherein the composition is a pharmaceutical composition.
 13. Acomposition comprising Trichophyton verrucosum strain DSM-28406.
 14. Avaccine formulation comprising Trichophyton verrucosum strain DSM-28406.15. A method of treating and/or preventing hoof- and claw diseases in ananimal or warts in a human comprising administering to said animal orhuman Trichophyton verrucosum DSM-28406.
 16. The method of claim 15,wherein the hoof- and claw disease is lameness, digital dermatitis,interdigital dermatitis and/or interdigital phlegmone, dermatophytosis.